Nematode PAN and ZP receptor-like sequences

ABSTRACT

Nucleic acid molecules from nematodes encoding PAN and ZP domain containing receptor polypeptides are described. PANZP polypeptide sequences are also provided, as are vectors, host cells, and recombinant methods for production of PANZP nucleotides and polypeptides. Also described are screening methods for identifying inhibitors and/or activators, as well as methods for antibody production.

RELATED APPLICATION INFORMATION

This application is a divisional of U.S. application Ser. No.10/771,708, filed Feb. 4, 2004, now U.S. Pat. No. 6,936,693 which claimspriority from U.S. provisional application Ser. No. 60/444,771, filedFeb. 4, 2003.

BACKGROUND

Nematodes (derived from the Greek word for thread) are active, flexible,elongate, organisms that live on moist surfaces or in liquidenvironments, including films of water within soil and moist tissueswithin other organisms. While only 20,000 species of nematode have beenidentified, it is estimated that 40,000 to 10 million actually exist.Some species of nematodes have evolved to be very successful parasitesof both plants and animals and are responsible for significant economiclosses in agriculture and livestock and for morbidity and mortality inhumans (Whitehead (1998) Plant Nematode Control, CAB International, NewYork).

Nematode parasites of plants can inhabit all parts of plants, includingroots, developing flower buds, leaves, and stems. Plant parasites areclassified on the basis of their feeding habits into the broadcategories: migratory ectoparasites, migratory endoparasites, andsedentary endoparasites. Sedentary endoparasites, which include the rootknot nematodes (Meloidogyne) and cyst nematodes (Globodera andHeterodera) induce feeding sites and establish long-term infectionswithin roots that are often very damaging to crops (Whitehead, supra).It is estimated that parasitic nematodes cost the horticulture andagriculture industries in excess of $78 billion worldwide a year, basedon an estimated average 12% annual loss spread across all major crops.For example, it is estimated that nematodes cause soybean losses ofapproximately $3.2 billion annually worldwide (Barker et al. (1994)Plant and Soil Nematodes: Societal Impact and Focus for the Future. TheCommittee on National Needs and Priorities in Nematology. CooperativeState Research Service, US Department of Agriculture and Society ofNematologists). Several factors make the need for safe and effectivenematode controls urgent. Continuing population growth, famines, andenvironmental degradation have heightened concern for the sustainabilityof agriculture, and new government regulations may prevent or severelyrestrict the use of many available agricultural anthelmintic agents.

The situation is particularly dire for high value crops such asstrawberries and tomatoes where chemicals have been used extensively tocontrol soil pests. The soil fumigant methyl bromide has been usedeffectively to reduce nematode infestations in a variety of thesespecialty crops. It is however regulated under the U.N. MontrealProtocol as an ozone-depleting substance and is scheduled forelimination in 2005 in the US (Carter (2001) Califonia Agriculture,55(3):2). It is expected that strawberry and other commodity cropindustries will be significantly impacted if a suitable replacement formethyl bromide is not found. Presently there are a very small array ofchemicals available to control nematodes and they are frequentlyinadequate, unsuitable, or too costly for some crops or soils (Becker(1999) Agricultural Research Magazine 47(3):22-24; U.S. Pat. No.6,048,714). The few available broad-spectrum nematicides such as Telone(a mixture of 1,3-dichloropropene and chloropicrin) have significantrestrictions on their use because of toxicological concerns (Carter(2001) California Agriculture, Vol. 55(3):12-18).

The macrocyclic lactones (e.g., avermectins and milbemycins) anddelta-toxins from Bacillus thuringiensis (Bt) are nematicidal activesthat in principle provide excellent specificity and efficacy and shouldallow environmentally safe control of plant parasitic nematodes.Unfortunately, in practice, these two approaches have proven lesseffective for agricultural applications against root pathogens. Althoughcertain avermectins show exquisite activity against plant parasiticnematodes these chemicals are hampered by poor bioavailability due totheir light sensitivity, degradation by soil microorganisms and tightbinding to soil particles (Lasota & Dybas (1990) Acta Leiden59(1-2):217-225; Wright & Perry (1998) Musculature and Neurobiology. In:The Physiology and Biochemistry of Free-Living and Plant-parasiticNematodes (eds R. N. Perry & D.J. Wright), CAB International 1998).Consequently despite years of research and extensive use against animalparasitic nematodes, mites and insects (plant and animal applications),macrocyclic lactones (e.g., avermectins and milbemycins) have never beencommercially developed to control plant parasitic nematodes in the soil.

Bt delta toxins must be ingested to affect their target organ, the brushborder of midgut epithelial cells (Marroquin et al. (2000) Genetics.155(4):1693-1699). Consequently they are not anticipated to be effectiveagainst the dispersal, non-feeding, juvenile stages of plant parasiticnematodes in the field. Because juvenile stages only commence feedingwhen a susceptible host has been infected, nematicides may need topenetrate the plant cuticle to be effective. In addition, soil mobilityof a relatively large 65-130 kDa protein—the size of typical Bt deltatoxins—is expected to be poor and transgenic delivery in planta islikely to be constrained by the exclusion of large particles by thefeeding tube of certain plant parasitic nematodes such as Heterodera(Atkinson et al. (1998) Engineering resistance to plant-parasiticnematodes. In: The Physiology and Biochemistry of Free-Living andPlant-parasitic Nematodes (eds R. N. Perry & D. J. Wright), CABInternational 1998).

Many plant species are known to be highly resistant to nematodes. Thebest documented of these include marigolds (Tagetes spp.), rattlebox(Crotalaria spectabilis), chrysanthemums (Chrysanthemum spp.), castorbean (Ricinus communis), margosa (Azardiracta indica), and many membersof the family Asteraceae (family Compositae) (Hackney & Dickerson.(1975) J Nematol 7(1):84-90). In the case of the Asteraceae, thephotodynamic compound alpha-terthienyl has been shown to account for thestrong nematicidal activity of the roots. Castor beans are plowed underas a green manure before a seed crop is set. However, a significantdrawback of the castor plant is that the seed contains toxic compounds(such as ricin) that can kill humans, pets, and livestock and is alsohighly allergenic. In many cases however, the active principle(s) forplant nematicidal activity has not been discovered and it remainsdifficult to derive commercially successful nematicidal products fromthese resistant plants or to transfer the resistance to agronomicallyimportant crops such as soybeans and cotton.

There remains an urgent need to develop environmentally safe,target-specific ways of controlling plant parasitic nematodes. In thespecialty crop markets, economic hardship resulting from nematodeinfestation is highest in strawberries, bananas, and other high valuevegetables and fruits. In the high-acreage crop markets, nematode damageis greatest in soybeans and cotton. There are however, dozens ofadditional crops that suffer from nematode infestation including potato,pepper, onion, citrus, coffee, sugarcane, greenhouse ornamentals andgolf course turf grasses.

Nematode parasites of vertebrates (e.g., humans, livestock and companionanimals) include gut roundworms, hookworms, pinworms, whipworms, andfilarial worms. They can be transmitted in a variety of ways, includingby water contamination, skin penetration, biting insects, or byingestion of contaminated food.

In domesticated animals, nematode control or “de-worming” is essentialto the economic viability of livestock producers and is a necessary partof veterinary care of companion animals. Parasitic nematodes causemortality in animals (e.g., heartworm in dogs and cats) and morbidity asa result of the parasites' inhibiting the ability of the infected animalto absorb nutrients. The parasite-induced nutrient deficiency leads todisease and stunted growth in livestock and companion animals; Forinstance, in cattle and dairy herds, a single untreated infection withthe brown stomach worm can permanently restrict an animal's ability toconvert feed into muscle mass or milk.

Two factors contribute to the need for novel anthelmintics and vaccinesfor control of parasitic nematodes of animals. First, some of the moreprevalent species of parasitic nematodes of livestock are buildingresistance to the anthelmintic drugs available currently, meaning thatthese products- will eventually lose their efficacy. These developmentsare not surprising because few effective anthelmintic drugs areavailable and most have been used continuously. Some parasitic specieshave developed resistance to most of the anthelmintics (Geents et al.(1997) Parasitology Today 13:149-151; Prichard (1994) VeterinaryParasitology 54:259-268). The fact that many of the anthelmintic drugshave similar modes of action complicates matters, as the loss ofsensitivity of the parasite to one drug is often accompanied by sideresistance—that is, resistance to other drugs in the same class(Sangster & Gill (1999) Parasitology Today 15(4):141-146). Secondly,there are some issues with toxicity for the major compounds currentlyavailable.

Infections by parasitic nematode worms result in substantial humanmortality and morbidity, especially in tropical regions of Africa, Asia,and the Americas. The World Health Organization estimates 2.9 billionpeople are infected, and in some areas, 85% of the population carriesworms. While mortality is rare in proportion to infections, morbidity issubstantial and rivals diabetes and lung cancer in worldwide disabilityadjusted life year (DALY) measurements.

Examples of human parasitic nematodes include hookworms, filarial worms,and pinworms. Hookworms (1.3 billion infections) are the major cause ofanemia in millions of children, resulting in growth retardation andimpaired cognitive development. Filarial worm species invade thelymphatics, resulting in permanently swollen and deformed limbs(elephantiasis), and the eyes, causing African river blindness. Thelarge gut roundworm Ascaris lumbricoides infects more than one billionpeople worldwide and causes malnutrition and obstructive bowel disease.In developed countries, pinworms are common and often transmittedthrough children in daycare.

Even in asymptomatic parasitic infections, nematodes can still deprivethe host of valuable nutrients and increase the ability of otherorganisms to establish secondary infections. In some cases, infectionscan cause debilitating illnesses and can result in anemia, diarrhea,dehydration, loss of appetite, or death.

Despite some advances in drug availability and public healthinfrastructure and the near elimination of one tropical nematode (thewater-borne Guinea worm), most nematode diseases have remainedintractable problems. Treatment of hookworm diseases with anthelminticdrugs, for instance, has not provided adequate control in regions ofhigh incidence because rapid re-infection occurs after treatment. Infact, over the last 50 years, while nematode infection rates have fallenin the United States, Europe, and Japan, the overall number ofinfections worldwide has kept pace with the growing world population.Large scale initiatives by regional governments, the World HealthOrganization, foundations, and pharmaceutical companies are now underwayattempting to control nematode infections with currently availabletools, including three programs for control of Onchocerciasis (riverblindness) in Africa and the Americas using ivermectin and vectorcontrol; The Global Alliance to Eliminate Lymphatic Filariasis usingDEC, albendazole, and ivermectin; and the highly successful Guinea WormEradication Program.

The obvious missing weapons in the fight to control human parasiticnematodes are vaccines. Systematic vaccination against childhooddiseases likes measles, mumps, polio, etc. has been among the mostimportant and cost effective factors increasing lifespan and wellness inthe developed world over the course of the 20th century. Expansion ofthese health gains into the developing world using existing vaccines, asthe Gates Foundation is supporting, has the potential to captureimmediate health gains. Such an approach could be equally effective fornematodes if such vaccines existed.

Research into vaccines for parasites, from malaria to nematode worms,has shown parasites to be challenging organisms to control byimmunization since, unlike many viruses, antibody or cellular responsesto most surface antigens fail to result in control. However, multiplevaccines for the control of nematode parasites in animals have shownefficacy either in testing or in veterinary use. For example,vaccination of dogs with irradiated hookworm larva results in highlevels of protection to subsequent hookworm challenge. The same approachworks for protection of gerbils from filarial worms. Unfortunately,parasitic nematodes cannot be grown in the quantities required for sucha killed whole organism vaccination approach, with limited exceptionssuch as the Intervet niche product HuskVac™ for cattle lungworm. Thegreatest commercial success to date in immunization for veterinaryparasites has come from the recombinant antigen vaccines TickGARD™ andGavac™ for cattle which block the lifecycle of the ectoparasiteBoophilus microplus, a bovine tick. Rather than utilizing a surfaceantigen, each of these vaccines targets an antigen, Bm86, expressed onthe luminal surface of the tick mid-gut so that as the ectoparasitedrinks the host's blood, it is exposed to antibodies that interfere withintestinal function. The same intestinal target approach has beensuccessful in small-scale trials against the sheep parasitic nematodeHaemonchus, a blood feeder similar to hookworms that can be controlledby vaccination with the purified parasite intestinal microvilli proteinH11. Importantly, unlike a typical vaccine where the antigen is used totrigger a cascade of immune attack on the entire organism, the parasiteintestinal approach utilizes an antibody response to “knockout” thefunction of a crucial nematode gene product, similar to the function ofa drug.

Finding effective compounds and vaccines against parasitic nematodes hasbeen complicated by the fact that the parasites have not been amenableto culturing in the laboratory. Parasitic nematodes are often obligateparasites (i.e., they can only complete their lifecycles in theirrespective hosts, such as in plants, animals, and/or humans) with slowgeneration times. Thus, they are difficult to grow under artificialconditions, making genetic and molecular experimentation difficult orimpossible. To circumvent these limitations, scientists have usedCaenorhabidits elegans as a model system for parasitic nematodediscovery efforts.

C. elegans is a small free-living bacteriovorous nematode that for manyyears has served as an important model system for multicellular animals(Burglin (1998) Int. J. Parasitol. 28(3):395-411). The genome of C.elegans has been completely sequenced and the nematode shares manygeneral developmental and basic cellular processes with vertebrates(Ruvkin et al. (1998) Science 282:2033-41). This, together with itsshort generation time and ease of culturing, has made it a model systemof choice for higher eukaryotes (Aboobaker et al. (2000) Ann. Med.32:23-30).

Although C. elegans serves as a good model system for vertebrates, it isan even better model for study of parasitic nematodes, as C. elegans andother nematodes share unique biological processes not found invertebrates. For example, unlike vertebrates, nematodes produce and usechitin, have gap junctions comprised of innexin rather than connexin andcontain glutamate-gated chloride channels rather than glycine-gatedchloride channels (Bargmann (1998) Science 282:2028-33). Thelatterproperty is of particular relevance given that the avermectinclass of drugs is thought to act at glutamate-gated chloride receptorsand is highly selective for invertebrates (Martin (1997) Vet. J.154:11-34).

A subset of the genes involved in nematode-specific processes will beconserved in nematodes and absent or significantly diverged fromhomologues in other phyla. In other words, it is expected that at leastsome of the genes associated with functions unique to nematodes willhave restricted phylogenetic distributions. The completion of the C.elegans genome project and the growing database of expressed sequencetags (ESTs) from numerous nematodes facilitate identification of these“nematode-specific” genes. In addition, conserved genes involved innematode-specific processes are expected to retain the same or verysimilar functions in different nematodes. This functional equivalencehas been demonstrated in some cases by transforming C. elegans withhomologous genes from other nematodes (Kwa et al. (1995) J. Mol. Biol.246:500-10; Redmond et al. (2001) Mol. Biochem. Parasitol. 112:125-131).This sort of data transfer has been shown in cross phyla comparisons forconserved genes and is expected to be more robust among species within aphylum. Consequently, C. elegans and other free-living nematode speciesare likely excellent surrogates for parasitic nematodes with respect toconserved nematode processes.

Many expressed genes in C. elegans and certain genes in otherfree-living nematodes can be “knocked out” genetically by a processreferred to as RNA interference (RNAi), a technique that provides apowerful experimental tool for the study of gene function in nematodes(Fire et al. (1998) Nature 391(6669):806-811; Montgomery et al. (1998)Proc. Natl. Acad Sci USA 95(26):15502-15507). Treatment of a nematodewith double-stranded RNA of a selected gene triggers the destruction ofexpressed sequences transcribed from that gene, thus reducing oreliminating expression of the corresponding protein. By preventing thetranslation of specific proteins, their functional significance andessentiality to the nematode can be assessed. Determination of essentialgenes and their corresponding proteins using C. elegans as a modelsystem will assist in the rational design of anti-parasitic nematodecontrol products.

SUMMARY

The invention features nucleic acid molecules encoding Strongyloidesstercoralis, Meloidogyne javanica, Heterodera glycines and Brugia malayiPANZP proteins, e.g., PANZP1 and PANZP2. S. stercoralis is a nematodeparasite that infects humans, primates, and dogs. It is one of the fewnematodes that can multiply within its host and can multiply uncheckedin immunosuppressed individuals. M. javanica is a Root Knot Nematodethat causes substantial damage to several crops, including cotton,tobacco, pepper, and tomato. H. glycines, referred to as Soybean CystNematode, is a major pest of soybean. B. malayi, is an arthropodvectored human parasite that is one of a causative agents of lymphaticfilariasis, a disease that afflicts roughly 120 million people worldwide. The PANZP proteins of the invention resemble the Drosophilamelanogaster no-mechanoreceptor potential A (nompA) and Sp71 proteins.The PANZP proteins of the invention include Plasminogen Apple Nematode(PAN) and Zona Pellucida (ZP) domains.

The PANZP nucleic acids and polypeptides of the invention allow for theidentification of nematode species. The nucleic acids and polypeptidesof the invention also allow for the identification of compounds thatbind to or alter the activity of PANZP polypeptides as well as compoundsthat alter the expression of PANZP polypeptides. Such compounds mayprovide a means for combating diseases and infestations caused bynematodes, particularly those caused by S. stercoralis, M. javanica, H.glycines and B. malayi (e.g., in mammals and plants). These nucleicacids and polypeptides also allow for the vaccination of animals andhumans against nematode parasites. In addition, anti-nematode peptide orprotein inhibitors and antibodies directed against nematode PAN and ZPcontaining proteins can be expressed in plants (plantibodies) to producetransgenic nematode resistance.

The invention is based, in part, on the identification of a cDNAencoding S. stercoralis PANZP1 (SEQ ID NO: 1). This 3750 nucleotide cDNAhas a 3369 nucleotide open reading frame (SEQ ID NO: 5) encoding an 1122amino acid polypeptide (SEQ ID NO: 3). The nucleotide and amino acidsequence of S. stercoralis PANZP1 is shown in FIGS. 1A-1C.

The invention is also based, in part, on the identification of a cDNAencoding S. stercoralis PANZP2 (SEQ ID NO: 2). This 1951 nucleotide cDNAhas a 1674 nucleotide open reading frame (SEQ ID NO: 6) encoding a 557amino acid polypeptide (SEQ ID NO: 4). The nucleotide and amino acidsequence of S. stercoralis PANZP2 is shown in FIGS. 2A-2B.

The invention is also based, in part, on the identification of a cDNAencoding M. javanica PANZP1 (SEQ ID NO: 7). This 3848 nucleotide cDNAhas a 3633 nucleotide open reading frame (SEQ ID NO: 13) encoding a 1210amino acid polypeptide (SEQ ID NO: 10). The nucleotide and amino acidsequence of M. javanica PANZP2 is shown in FIGS. 3A-3C.

The invention is also based, in part, on the identification of a partialcDNA fragment encoding H. glycines PANZP1 (SEQ ID NO: 8). This 752nucleotide partial cDNA fragment has a 750 nucleotide open reading frame(SEQ ID NO: 14) encoding a 250 amino acid polypeptide (SEQ ID NO: 11).The nucleotide and amino acid sequence of H. glycines PANZP2 is shown inFIG. 4.

The invention is also based, in part, on the identification of a partialcDNA fragment encoding B. malayi PANZP1 (SEQ ID NO: 9). This 2808nucleotide partial cDNA fragment has a 2643 nucleotide open readingframe (SEQ ID NO: 15) encoding a 881 amino acid polypeptide (SEQ ID NO:12). The nucleotide and amino acid sequence of B. malayi PANZP2 is shownin FIGS. 5A-5B.

In one aspect, the invention features novel nematode PAN and ZPcontaining receptor-like polypeptides. Such polypeptides includepurified polypeptides having the amino acid sequences set forth in SEQID NO: 3, 4, 10, 11 and/or 12. Also included are polypeptides having anamino acid sequence that is at least about 80%, 85%, 90%, 95%, or 98%identical to SEQ ID NO: 3, 4, 10, 11 and/or 12 as well as polypeptideshaving a sequence that differs from that of SEQ ID NO: 3, 4, 10, 11and/or 12 at 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 residues (aminoacids). The purified polypeptides can be encoded by a nematode gene,e.g., a nematode gene other than a C. elegans gene. For example, thepurified polypeptide has a sequence other than SEQ ID NO: 16, 17, and 18(C. elegans PANZP1 and PANZP2 proteins). The purified polypeptides canfurther include a heterologous amino acid sequence, e.g., anamino-terminal or carboxy-terminal sequence. Also featured are purifiedpolypeptide fragments of the aforementioned PANZP polypeptides, e.g., afragment of at least about 20, 30, 40, 50, 75, 85, 104, 106, 113 150,200, 250 amino acids. Non-limiting examples of such fragments include:fragments from about amino acid 20 to 110 and 100 to 210 of SEQ ID NO:3, 4, 10, 11 and/or 12 and 200 to 310, 300 to 400 and 400 to 500 of SEQID NO: 3, 4, 10 and/or 12. The polypeptide or fragment thereof can bemodified, e.g., processed, truncated, modified (e.g. by glycosylation,phosphorylation, acetylation, myristylation, prenylation,palmitoylation, amidation, addition of glycerophosphatidyl inositol), orany combination of the above. Certain PANZP polypeptides comprise asequence of 600, 700, 800, 900, 1000, 1100, 1200, 1300 amino acids orfewer. The invention also features polypeptides comprising, consistingessentially of or consisting of the aforementioned polypeptides. Alsowithin the invention are polypeptides, including immunogenicpolypeptides comprising (or consisting of or consisting essentially of)a PAN or ZP domain of SEQ ID NO: 3, 4, 10, 11, or 12, e.g., a PAN or ZPdomain listed in Table 3.

In another aspect, the invention features novel isolated nucleic acidmolecules encoding nematode PAN and ZP containing receptor-likepolypeptides. Such isolated nucleic acid molecules include nucleic acidshaving the nucleotide sequence set forth in SEQ ID NO: 1, 2, 7, 8 or SEQID NO: 9. Also included are isolated nucleic acid molecules having thesame sequence as or encoding the same polypeptide as a nematode PAN andZP containing receptor-like gene (other than a C. elegans PANZP genes).

Also featured are: 1) isolated nucleic acid molecules having a strandthat hybridizes under low stringency conditions to a single strandedprobe of the sequences of SEQ ID NO: 1, 2, 7, 8 , 9, or theircomplements and, optionally, encodes polypeptides of between 500 and1300 amino acids; 2) isolated nucleic acid molecules having a strandthat hybridizes under high stringency conditions to a single strandedprobe of the sequence of SEQ ID NO: 1, 2, 7, 8, 9 or their complementsand, optionally, encodes polypeptides of between 500 and 1300 aminoacids; 3) isolated nucleic acid fragments of a PANZP nucleic acidmolecule, e.g., a fragment of SEQ ID NO: 1, 2, 7, 8 or 9 that is about50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 1000, 1500, 2000, 2500,3000 and 3500 or more nucleotides in length or ranges between suchlengths; and 4) oligonucleotides that are complementary to a PANZPnucleic acid molecule or a PANZP nucleic acid complement, e.g., anoligonucleotide or probe of about 10, 15, 18, 20, 22, 24, 28, 30, 35,40, 50, 60, 70, 80, or more nucleotides in length. Exemplaryoligonucleotides are oligonucleotides which anneal to a site locatedbetween nucleotides about 1 to 96, 1 to 180, 1 to 270, 1 to 324, 96 to324, 96 to 345, 324 to 345, 324 to 603, 345 to 603, 345 to 618, 603 to618, 603 to 752 of SEQ ID NO: 1, 2, 7, 8 or 9; 618 to 1197, 906 to 1524,1524 to 1951 of SEQ ID NO: 1, 2, 7 or 9; 1197 to 2124, 1524 to 2808 ofSEQ ID NO: 1, 7, or 9; 1197 to 2124, 1524 to 3750 of SEQ ID NO: 1 or 7;1197 to 2124, 1524 to 3848 of SEQ ID NO: 7. Nucleic acid fragmentsinclude the following non-limiting examples: nucleotides about 1 to 200of SEQ ID NO: 1, 2, 7, 8, or 9, 100 to 300, 200 to 400, 300 to 500, 400to 700, 500 to 800, 600 to 1200, 1200 to 1951 of SEQ ID NO: 1, 2, 7, or9, 1200 to 2808 of SEQ ID NO: 1, 7 or 9; 1200 to 3750 of SEQ ID NO: 1 or7, 1200 to 3848 of SEQ ID NO: 7. Also within the invention are nucleicacid molecules that hybridize under stringent conditions to nucleic acidmolecule comprising SEQ ID NO: 1, 2, 7, 8 or 9 and comprise 4000, 3000,2000, 1000 or fewer nucleotides. The isolated nucleic acid can furtherinclude a heterologous promoter or other sequences required fortranscription or translation of the nucleic acid molecule in a cell,e.g., a mammalian or eukaryotic or prokaryotic cell, operably linked tothe PANZP nucleic acid molecule. The isolated nucleic acid molecule canencode a polypeptide having PAN and ZP containing receptor-likefunction.

A molecule-featured herein can be from a nematode of the classAraeolaimida, Ascaridida, Chromadorida, Desmodorida, Diplogasterida,Monhysterida, Mononchida, Oxyurida, Rhigonematida, Spirurida, Enoplia,Desmoscolecidae, Rhabditida, or Tylenchida. Alternatively, the moleculecan be from a species of the class Rhabditida, particularly a speciesother than C. elegans.

In another aspect, the invention features a vector, e.g., a vectorcontaining an aforementioned nucleic acid. The vector can furtherinclude one or more regulatory elements, e.g., a heterologous promoteror elements required for translation. The regulatory elements fordirecting transcription and translation elements can be suitable forexpression in bacteria, plants, animals, or insects. The regulatoryelements can be operably linked to the PAN and ZP containingreceptor-like nucleic acid molecules in order to express a PANZP nucleicacid molecule. In yet another aspect, the invention features atransgenic cell or transgenic organism having in its genome a transgenecontaining an aforementioned PANZP nucleic acid molecule and aheterologous nucleic acid, e.g., a heterologous promoter.

In still another aspect, the invention features an antibody, e.g., anantibody, antibody fragment, or derivative thereof that bindsspecifically to an aforementioned polypeptide. Such antibodies can bepolyclonal or monoclonal antibodies. The antibodies can be modified,e.g., humanized, rearranged as a single-chain, or CDR-grafted. Theantibodies may be directed against a fragment, a peptide, or adiscontinuous epitope from a PANZP polypeptide. The antibody need notinclude domain that trigger an immune response.

In another aspect, the invention features a method of screening for acompound that binds to a nematode PANZP polypeptide, e.g., anaforementioned polypeptide. The method includes providing the nematodepolypeptide; contacting a test compound to the polypeptide; anddetecting binding of the test compound to the nematode polypeptide. Inone embodiment, the method further includes contacting the test compoundto a mammalian PAN or ZP domain-containing polypeptide and detectingbinding of the test compound to the mammalian PAN or ZP polypeptide inorder to identify compounds with selective binding activity. A testcompound that binds the nematode PANZP polypeptide with at least 2-fold,5-fold, 10-fold, 20-fold, 50-fold, or 100-fold affinity greater relativeto its affinity for the mammalian (e.g., a human) PAN or ZP polypeptidecan be identified.

The invention also features methods for identifying compounds that alter(increases or decreases) the association of a nematode PAN and ZPcontaining domain receptor-like polypeptide with a substrate such as asmall molecule or protein. The method includes contacting the testcompound to the nematode PANZP polypeptide; and detecting a decrease inthe binding of the PANZP protein to the substrate. A decrease in thelevel of PANZP polypeptide binding to the substrate relative to thePANZP polypeptide binding to the substrate in the absence of the testcompound is an indication that the test compound is an inhibitor of thePANZP activity. The inhibitor can be a direct competitor of the bindingor an allosteric inhibitor that prevents binding of the PANZPpolypeptide to other molecules or proteins. Such inhibitory compoundsare potential selective agents for reducing the viability of a nematodeexpressing a PANZP polypeptide, e.g., S. stercoralis, M. javanica, H.glycines and B. malayi. These methods can also include contacting thecompound with a vertebrate PAN containing protein (e.g., human FactorXI) or a vertebrate ZP containing polypeptide (e.g., human uromodulin)and detecting binding of the compounds to the proteins. A compound thatbinds to the nematode PAN and ZP containing receptor-like polypeptidesto a greater extent than it binds to vertebrate PAN or ZP polypeptidescould be useful as a selective inhibitor of the nematode polypeptide. Adesirable compound can exhibit 2-fold, 5-fold, 10-fold, 20-fold,50-fold, 100-fold or greater selective affinity against the nematodepolypeptide.

Another featured method is a method of screening for a compound thatalters (increases or. decreases) the binding of a PAN and ZP containingreceptor-like polypeptide to a small molecule or protein substrate oralters the regulation of other polypeptides by the PANZP protein. Themethod includes providing the PANZP polypeptide; contacting a testcompound to the PANZP polypeptide; and detecting an alteration of thebinding activity or the activity of polypeptides regulated by the .PANZPprotein, wherein a change in binding activity of the PANZP polypeptidesto its substrates or a change in the activity of other polypeptidesdownstream of the PANZP protein binding activity relative to the bindingactivity of the PANZP protein or the activity of downstream polypeptidesin the absence of the test compound is an indication that the testcompound alters the activity of the PANZP polypeptide(s). The method canfurther include contacting the test compound to a vertebrate PANcontaining protein (e.g., human Factor XI) or a vertebrate ZP containingpolypeptide (e.g., human uromodulin) and detecting binding of thecompounds to the proteins and measuring the effects of the compounds onthe activities of the vertebrate proteins. A test compound that altersthe activity of the nematode PANZP polypeptide at a given concentrationand that does not substantially alter the activity of the vertebrate PANor ZP containing polypeptide or downstream polypeptides at the givenconcentration can be identified. An additional method includes screeningfor both binding to a PANZP polypeptide and for an alteration in thebinding activity of a PANZP polypeptide. Yet another featured method isa method of screening for a compound that alters (increases ordecreases) the viability or fitness of a transgenic cell or organism ornematode. The transgenic cell or organism has a transgene that expressesa PAN and ZP containing receptor-like polypeptide. The method includescontacting a test compound to the transgenic cell or organism anddetecting changes in the viability or fitness of the transgenic cell ororganism. This alteration in viability or fitness can be measuredrelative to an otherwise identical cell or organism that does not harborthe transgene.

Also featured is a method of screening for a compound that alters theexpression of a nematode nucleic acid encoding a PAN and ZP containingreceptor-like polypeptide, e.g., a nucleic acid encoding a S.stercoralis, M. javanica, H. glycines or B. malayi PANZP polypeptide.The method includes contacting a cell, e.g., a nematode cell, with atest compound and detecting expression of a nematode nucleic acidencoding a PANZP polypeptide, e.g., by hybridization to a probecomplementary to the nematode nucleic acid encoding a PANZP polypeptideor by contacting polypeptides isolated from the cell with a compound,e.g., antibody that binds a PANZP polypeptide. Compounds identified bythe method are also within the scope of the invention.

In yet another aspect, the invention features a method of treating adisorder (e.g., an infection) caused by a nematode, e.g., S.stercoralis, M javanica, H. glycines or B. malayi in a subject, e.g., ahost plant or host animal. The method includes administering to thesubject an effective amount of an inhibitor of a PANZP polypeptideactivity or an inhibitor of expression of a PANZP polypeptide.Non-limiting examples of such inhibitors include: an antisense nucleicacid (or PNA) to a PANZP nucleic acid, a double-stranded RNA inhibitorcapable of triggering RNA interference, an antibody to a PANZPpolypeptide, an inhibitory peptide or protein, or a small moleculeidentified as a PANZP polypeptide inhibitor by a method describedherein.

Also featured is a method of preventing or treating a disorder (e.g., aninfection) caused by a nematode (e.g., S. stercoralis or B. malayi) in ahost animal by vaccinating the animal with nematode PANZP protein ornucleic acid (e.g., a PANZP DNA vaccine) or both. Also featured is amethod of preventing infection of a plant host by a nematode (e.g., M.javanica or H. glycines) by expressing an antisense RNA ordouble-stranded RNA to the nematode PANZP nucleic acid or by expressingantibodies or other proteins which interfere with the function of thenematode PANZP protein.

In yet another aspect, the invention features methods for the productionof nematode resistant transgenic plants by obtaining specific antibodiesto nematode PANZP proteins, deriving the nucleic acid sequences thatcode for these antibodies and expressing these nucleic acids in plantsunder the control of appropriate promoters (e.g., constitutive orinducible, non-tissue specific, root specific, feeding site specific)and with other suitable control sequences (e.g., enhancers, introns,UTRs, terminators) to produce antibodies to the PANZP proteins in plants(plantibodies).

Also featured in this invention is a method of producing nematoderesistant transgenic plants by the expression of nucleic acids codingfor PANZP nematode proteins or portions of PANZP nematode proteins thatcan produce PANZP peptides or polypeptides capable of dominant negativeinteraction with endogenous nematode PAN and ZP containing receptor-likeproteins upon ingestion by plant parasitic nematodes.

Also within the scope of this invention is the use of selectiontechniques like phage display or polysome display to generate peptidesor proteins which bind to and inhibit the function of nematode PANZPproteins.

A “purified polypeptide”, as used herein, refers to a polypeptide thathas been separated from other proteins, lipids, and nucleic acids withwhich it is naturally associated. The polypeptide can constitute atleast 10, 20, 50 70, 80 or 95% by dry weight of the purifiedpreparation.

An “isolated nucleic acid” is a nucleic acid, the structure of which isnot identical to that of any naturally occurring nucleic acid, or tothat of any fragment of a naturally occurring genomic nucleic acidspanning more than three separate genes. The term therefore covers, forexample: (a) a DNA which is part of a naturally occurring genomic DNAmolecule but is not flanked by both of the nucleic acid sequences thatflank that part of the molecule in the genome of the organism in whichit naturally occurs; (b) a nucleic acid incorporated into a vector orinto the genomic DNA of a prokaryote or eukaryote in a manner such thatthe resulting molecule is not identical to any naturally occurringvector or genomic DNA; (c) a separate molecule such as a cDNA, a genomicfragment, a fragment produced by polymerase chain reaction (PCR), or arestriction fragment; and (d) a recombinant nucleotide sequence that ispart of a hybrid gene, i.e., a gene encoding a fusion protein.Specifically excluded from this definition are nucleic acids present inmixtures of different (i) DNA molecules, (ii) transfected cells, or(iii) cell clones in a DNA library such as a cDNA or genomic DNAlibrary. Isolated nucleic acid molecules according to the presentinvention farther include molecules produced synthetically, as well asany nucleic acids that have been altered chemically and/or that havemodified backbones.

Although the phrase “nucleic acid molecule” primarily refers to thephysical nucleic acid molecule and the phrase “nucleic acid sequence”refers to the sequence of the nucleotides in the nucleic acid molecule,the two phrases can be used interchangeably.

The term “substantially pure” as used herein in reference to a givenpolypeptide means that the polypeptide is substantially free from otherbiological macromolecules. The substantially pure polypeptide is atleast 75% (e.g., at least 80, 85, 95, or 99%) pure by dry weight. Puritycan be measured by any appropriate standard method, for example, bycolumn chromatography, polyacrylamide gel electrophoresis, or HPLCanalysis.

A percent identity for any subject nucleic acid or amino acid sequence(e.g., any of the PANZP polypeptides described herein) relative toanother “target” nucleic acid or amino acid sequence can be determinedas follows. First, a target nucleic acid or amino acid sequence of theinvention can be compared and aligned to a subject nucleic acid or aminoacid sequence, using the BLAST 2 Sequences (B12seq) program from thestand-alone version of BLASTZ containing BLASTN and BLASTP (e.g.,version 2.0.14). The stand-alone version of BLASTZ can be obtained at orwww.ncbi.nlm.nih.gov>. Instructions explaining how to use BLASTZ, andspecifically the B12seq program, can be found in the ‘readme’ fileaccompanying BLASTZ. The programs also are described in detail by Karlinet al. (1990) Proc. Natl. Acad. Sci. 87:2264; Karlin et al. (1990) Proc.Natl. Acad. Sci. 90:5873; and Altschul et al. (1997) Nucl. Acids Res.25:3389.

B12seq performs a comparison between the subject sequence and a targetsequence using either the BLASTN (used to compare nucleic acidsequences) or BLASTP (used to compare amino acid sequences) algorithm.Typically, the default parameters of a BLOSUM62 scoring matrix, gapexistence cost of 11 and extension cost of 1, a word size of 3, anexpect value of 10, a per residue cost of 1 and a lambda ratio of 0.85are used when performing amino acid sequence alignments. The output filecontains aligned regions, of homology between the target sequence andthe subject sequence. Once aligned, a length is determined by countingthe number of consecutive nucleotides or amino acid residues (i.e.,excluding gaps) from the target sequence that align with sequence fromthe subject sequence starting with any matched position and ending withany other matched position. A matched position is any position where anidentical nucleotide or amino acid residue is present in both the targetand subject sequence. Gaps of one or more residues can be inserted intoa target or subject sequence to maximize sequence alignments betweenstructurally conserved domains (e.g., α-helices, β-sheets, and loops).

The percent identity over a particular length is determined by countingthe number of matched positions over that particular length, dividingthat number by the length and multiplying the resulting value by 100.For example, if (i) a 500 amino acid target sequence is compared to asubject amino acid sequence, (ii) the B12seq program presents 200 aminoacids from the target sequence aligned with a region of the subjectsequence where the first and last amino acids of that 200 amino acidregion are matches, and (iii) the number of matches over those 200aligned amino acids is 180, then the 500 amino acid target sequencecontains a length of 200 and a sequence identity over that length of 90%(i.e., 180÷200×100=90).

It will be appreciated that a nucleic acid or amino acid target sequencethat aligns with a subject sequence can result in many different lengthswith each length having its own percent identity. It is noted that thepercent identity value can be rounded to the nearest tenth. For example,78.11, 78.12, 78.13, and 78.14 is rounded down to 78.1, while 78.15,78.16, 78.17, 78.18, and 78.19 is rounded up to 78.2. It is also notedthat the length value will always be an integer.

The identification of conserved regions in a template, or subject,polypeptide can facilitate homologous polypeptide sequence analysis.Conserved regions can be identified by locating a region within theprimary amino acid sequence of a template polypeptide that is a repeatedsequence, forms some secondary structure (e.g., helices and betasheets), establishes positively or negatively charged domains, orrepresents a protein motif or domain. See, e.g., the Pfam web sitedescribing consensus sequences for a variety of protein motifs anddomains at http://www.sanger.ac.uk/Pfam/ andhttp://genome.wustl.edu/Pfam/. A description of the information includedat the Pfam database is described in Sonnhammer et al. (1998) Nucl.Acids Res. 26: 320-322; Sonnhammer et al. (1997) Proteins 28:405-420;and Bateman et al. (1999) Nucl. Acids Res. 27:260-262. From the Pfamdatabase, consensus sequences of protein motifs and domains can bealigned with the template polypeptide sequence to determine conservedregion(s).

As used herein, the term “transgene” means a nucleic acid sequence(encoding, e.g., one or more subject polypeptides), which is partly orentirely heterologous, i.e., foreign, to the transgenic plant, animal,or cell into which it is introduced, or, is homologous to an endogenousgene of the transgenic plant, animal, or cell into which it isintroduced, but which is designed to be inserted, or is inserted, intothe plant's genome in such a way as to alter the genome of the cell intowhich it is inserted (e.g., it is inserted at a location which differsfrom that of the natural gene or its insertion results in a knockout). Atransgene can include one or more transcriptional regulatory sequencesand other nucleic acid sequences, such as introns, that may be necessaryfor optimal expression of the selected nucleic acid, all operably linkedto the selected nucleic acid, and may include an enhancer sequence.

As used herein, the term “transgenic cell” refers to a cell containing atransgene.

As used herein, a “transgenic plant” is any plant in which one or more,or all, of the cells of the plant includes a transgene. The transgenecan be introduced into the cell, directly or indirectly by introductioninto a precursor of the cell, by way of deliberate genetic manipulation,such as by T-DNA mediated transfer, electroporation, or protoplasttransformation. The transgene may be integrated within a chromosome, orit may be extrachromosomally replicating DNA.

As used herein, the term “tissue-specific promoter” means a DNA sequencethat serves as a promoter, i.e., regulates expression of a selected DNAsequence operably linked to the promoter, and which affects expressionof the selected DNA sequence in specific cells of a tissue, such as aleaf, root, or stem.

As used herein, the terms “hybridizes under stringent conditions” and“hybridizes under high stringency conditions” refer to conditions forhybridization in 6× sodium chloride/sodium citrate (SSC) buffer at about45° C., followed by two washes in 0.2×SSC buffer, 0.1% SDS at 60° C. or65° C. As used herein, the term “hybridizes under low stringencyconditions” refers to conditions for hybridization in 6×SSC buffer atabout 45° C., followed by two washes in 6×SSC buffer, 0.1% (w/v) SDS at50° C.

A “heterologous promoter”, when operably linked to a nucleic acidsequence, refers to a promoter which is not naturally associated withthe nucleic acid sequence.

As used herein, an agent with “anthelmintic activity” is an agent, whichwhen tested, has measurable nematode-killing activity or results ininfertility or sterility in the nematodes such that unviable or nooffspring result. In the assay, the agent is combined with nematodes,e.g., in a well of microtiter dish having agar media or in the soilcontaining the agent. Staged adult nematodes are placed on the media.The time of survival, viability or number of offspring, and/or themovement of the nematodes are measured. An agent with “anthelminticactivity” reduces the survival time of adult nematodes relative tounexposed similarly staged adults, e.g., by about 20%, 40%, 60%, 80%, ormore. In the alternative, an agent with “anthelmintic activity” may alsocause the nematodes to cease replicating, regenerating, and/or producingviable progeny, e.g., by about 20%, 40%, 60%, 80%, or more.

As used herein, the term “binding” refers to the ability of a firstcompound and a second compound that are not covalently linked tophysically interact. The apparent dissociation constant for a bindingevent can be 1 mM or less, for example, 10 nM, 1 nM, 0.1 nM or less.

As used herein, the term “binds specifically” refers to the ability ofan antibody to discriminate between a target ligand and a non-targetligand such that the antibody binds to the target ligand and not to thenon-target ligand when simultaneously exposed to both the given ligandand non-target ligand, and when the target ligand and the non-targetligand are both present in molar excess over the antibody.

As used herein, the term “altering an activity” refers to a change inlevel, either an increase or a decrease in the activity, (e.g., anincrease or decrease in the ability of the polypeptide to bind orregulate other polypeptides or molecules) particularly a PANZP activity.The change can be detected in a qualitative or quantitative observation.If a quantitative observation is made, and if a comprehensive analysisis performed over a plurality of observations, one skilled in the artcan apply routine statistical analysis to identify modulations where alevel is changed and where the statistical parameter, the p value, isless than 0.05.

In part, the nematode PAN and ZP containing receptor-like proteins andnucleic acids described herein are novel targets for anti-nematodevaccines, pesticides, and drugs. These polypeptides are also useful forthe creation of nematode resistant transgenic plants. Inhibition ofthese molecules can provide means of inhibiting nematode metabolismand/or the nematode life-cycle.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A-1C depict the cDNA sequence of a S. stercoralis PAN and ZPcontaining receptor-like protein 1 (PANZP1) (SEQ ID NO: 1), itscorresponding encoded amino acid sequence (SEQ ID NO: 3), and its openreading frame (SEQ ID NO: 5).

FIGS. 2A-2B depict the cDNA sequence of a S. stercoralis PAN and ZPcontaining receptor-like protein 2 (PANZP2) (SEQ ID NO: 2), itscorresponding encoded amino acid sequence (SEQ ID NO: 4), and its openreading frame (SEQ ID NO: 6).

FIGS. 3A-3C depict the cDNA sequence of a M. javanica PAN and ZPcontaining receptor-like protein 1 (PANZP1) (SEQ ID NO: 7), itscorresponding encoded amino acid sequence (SEQ ID NO: 10), and its openreading frame (SEQ ID NO: 13).

FIG. 4 depicts the partial cDNA fragment of the sequence of a H.glycines PAN and ZP containing receptor-like protein 1 (PANZP1) (SEQ IDNO: 8), its corresponding encoded amino acid sequence (SEQ ID NO: 11),and its open reading frame (SEQ ID NO: 14).

FIGS. 5A-5B depict the partial cDNA fragment of a sequence of a B.malayi PAN and ZP containing receptor-like protein 1 (PANZP1) (SEQ IDNO: 9), its corresponding encoded amino acid sequence (SEQ ID NO: 12),and its open reading frame (SEQ ID NO: 15).

FIG. 6 depicts an alignment showing schematic depictions of a number ofPAN and ZP domain containing proteins including: D. melanogasterGenBank® Accession No. NP_(—)524831, C. elegans GenBank® Accession No.NP_(—)505875, C. elegans GENBANK® Accession No. NP_(—)502253, C. elegansGENBANK® Accession No. NP_(—)505874, C. elegans GENBANK® Accession No.NP_(—)502699, C. elegans GENBANK® Accession No. NP_(—)501670, C. elegansGENBANK® Accession No. NP 502252, C. elegans GENBANK® Accession No.NP_(—)491706, C. elegans GENBANK® Accession No. AAB52479, D.melanogaster GENBANK® Accession No. AAK09434.

FIG. 7 is an alignment of the sequences of S. stercoralis, M. javanica,H. glycines and B. malayi PAN and ZP containing receptor-likepolypeptide proteins and fragments (PANZP1; SEQ ID NO: 3, 10, 11, 12),C. elegans PANZP1 polypeptides (SEQ ID NO: 16 and 17) and C. briggsaePANZP1 polypeptide (SEQ ID NO: 45).

FIG. 8 is an alignment of the sequences of S. stercoralis PAN and ZPcontaining receptor-like polypeptide protein 2 (PANZP2; SEQ ID NO: 4),C. elegans PANZP2 polypeptide (SEQ ID NO: 18) and C. briggsae PANZP2(SEQ ID NO: 46).

DETAILED DESCRIPTION

An important step toward the development of new anthelmintic agents isthe identification of nematode-specific gene products that can serve astargets for inhibitory peptides and proteins (e.g., antibodies) andantiparasitic chemicals. An ideal target gene would be essential fornematode viability, such that interference with the target would resultin the arrest of parasite growth and reproduction. In addition, theprotein product of the target gene should be accessible to drugs, smallchemicals or antibodies. Finally, the ideal target should be specific tonematodes and not closely related to any gene in plants or animals.Based on these criteria, we have identified two C. elegans genes, PANZP1(C34G6.6) and PANZP2 (F52B11.3), as important targets for thedevelopment of vaccines, small molecule anthelmintic chemicals for bothhuman and animal parasites and nematicides for plant parasitic nematodecontrol. In addition, inhibitors of PANZP1 and PANZP2 could be used inthe design of transgenic plants producing anti-nematode peptides, smallnatural products with nematicidal activity, and antibodies(plantibodies) directed against endogenous nematode targets.

PANZP1 and PANZP2 are predicted to be secreted, membrane-bound proteins.Close homologs of the C. elegans PANZP1 genes were identified in anintestinal library from Ascaris suum, suggesting that these genes areexpressed in the nematode gastrointestinal system. The presence ofC-terminal transmembrane domains suggests that the proteins are anchoredin the membrane. Domain analysis of the PANZP1 and PANZP2 sequencesusing TargetP (a secretion prediction tool available on the internet atcbs.dtu.dk/services/TargetP/), PFAM (a domain analysis tool available onthe internet at pfam.wustl.edu;) and TMHMM (a transmembrane domainprediction tool available on the internet at cbs.dtu.dk/services/TMHMM)indicates the presence of a secretion leader, several Plasminogen AppleNematode (PAN) domains and a single C-terminal Zona Pellucida (ZP)domain before the transmembrane helix which is followed by a shortC-terminal tail. The domain architecture of PANZP1 and PANZP2 isillustrated at the top of FIG. 6 along with the domain structure of anumber of C. elegans and D. melanogaster PAN and ZP domain-containingproteins. The C. elegans genome contains several predicted proteins witha similar modular arrangement to PANZP1 and PANZP2. Of these, two(C34G6.6a and C34G6.6b) appear to be isoforms of the same gene(C34G6.6[p] is an older gene prediction), but the others appear torepresent unique loci. Additionally, homologs containing the same domainlayout are found in Drosophila melanogaster and the Anopheles gambiaegenomes.

PAN domains and the related PAN_AP domains are typically 80-90 aminoacids in length and are defined by a characteristic pattern of sixcysteine residues and conserved hydrophobic residues. The cysteineresidues form three highly conserved disulfide bonds linking the firstand sixth, second and fifth, and third and fourth cysteine residuespresent in each repeat (McMullen et al. (1991) Biochemistry,30(8):2050-2056; Brown et al. (2001) FEBS Lett. 497(1)31-38). Theconserved disulfide linkages give the PAN domains a characteristicapple-like globular structure. PAN domains were originally referred toas “Apple” domains based on this characteristic structure.

PAN and PAN_AP domains have been extensively studied in the mammalianblood coagulation proteins Factor XI (FXI), plasma pre-kallikrein (PK),and plasminogen. The specific involvement of the apple (or PAN) domainsin protein-protein interactions that mediate blood clotting has beendemonstrated (Baglia et al. (1995) J. Biol. Chem. 270(12):6734-6740; Sun& Gailani (1996) J. Biol. Chem. 271(46):29023-29028; Ho et al. (2000)Biochemistry, 39(2):316-323; Renne et al. (2002) J. Biol. Chem.277(7):4892-4899). PAN domains are also thought to mediateprotein-protein or protein-carbohydrate interactions in adhesiveproteins that are secreted by apicomplexan parasites, single-celledeukaryotic organisms that invade target host cells in order toreplicate. PAN domain-containing proteins are secreted by theseorganisms and are thought to play a role in the recognition andattachment of the parasite to host cells (Brown et al. (2001) FEBS Lett.497(1)31-38; Brecht et al. (2001) J. Biol. Chem. 276(6):4119-4127).

The C. elegans genome contains at least 20 predicted proteins thatcontain one or more PAN domains. Although the level of sequence percentidentity is low among the PAN domain family members, the pattern ofconserved cysteines and hydrophobic residues establishes the threedimensional structure that is characteristic of the domain (Tordai etal. (1999) FEBS Lett. 461(1-2):63-67). The possibility for a high degreeof sequence diversity within the family enables the domain to mediate alarge number of protein-protein interactions.

In addition to N-terminal PAN domains, PANZP1 and PANZP2 contain aC-terminal ZP (zona pellucida) domain. Many eukaryotic proteins containZP domains, including the mammalian sperm cell receptors ZP2 and ZP3 andother large modular transmembrane proteins such as the major urinaryprotein uromodulin (Tamm-Horsfall protein or THP), human alpha-tectorin,and Drosophila nompA. In all examples found to date, the ZP domainoccurs at the C-terminus of the protein.

The ZP domain occurs in proteins that are known to polymerize to formfilaments and matrices. For example, THP, the most abundant urinaryprotein, is a secreted protein that polymerizes into filaments that arethought to be responsible for the water-impermeability of the thickascending limb of the loop of Henle (Kokot & Dulawa (2000) Nephron,85(2):97-102). Mammalian sperm receptors ZP2 and ZP3 are secreted byoocytes and polymerize to form the thick extracellular matrix, the zonapellucida, which surrounds oocytes. Another ZP domain-containingprotein, alpha-tectorin, is the primary non-collagenous component of thecochlear tectorial membrane, an extracellular matrix that is importantin the transduction of sound into neuronal impulses. The requirement ofthe ZP domain for the assembly of THP and for ZP2 and ZP3 proteins intosupramolecular filaments was recently demonstrated (Jovine et al. (2002)Nat. Cell. Biol. 4(6):457-461).

The Drosophila nompA gene has a similar domain arrangement to PANZP1 andPANZP2, and while overall sequence percent identity between the insectand nematode proteins is low, the nompA protein (along with Sp71) is oneof the most closely related non-nematode sequences by BLAST analysis to(the C. elegans PAN-ZP containing proteins). Like PANZP1 and PANZP2, theDrosophila nompA (no-mechanoreceptor potential A) is a transmembraneprotein with a large, modular extracellular segment that includes thePAN and ZP domains. NompA is localized in an extracellular matrix thatis responsible for the transduction of mechanical stimuli to sensoryprocesses in the peripheral nervous system (Chung et al. (2001) Neuron29(2):415-428). Mutations in the no-mechanoreceptor-potential A (nompA)gene eliminate transduction in Drosophila mechanosensory organs bydisrupting contacts between neuronal sensory endings and cuticularstructures.

PANZP1 and PANZP2 are essential for nematode viability. RNAi-generatedmutations of PANZP1 and PANZP2 result in larval arrest at the L2 stage.A related C. elegans PAN-domain containing protein, LET-653 (C29E6.1)has also been shown to be an essential gene (Clark & Baille (1992) Mol.Gen. Genet. 232(1):97-105). Mutations in the let-653 gene are lethal andare associated with the appearance of large vacuoles that suggest adysfunction of the secretory/excretory apparatus (Jones & Baille (1995)Mol. Gen. Genet. 248(6):719-726). LET-653 has two N-terminal PAN domainsand a weakly predicted C-terminal ZP domain that contains a region oflow-complexity sequence. The function of LET-653 is unknown, but it hasbeen speculated that it may be functionally similar to the mammalianZP-domain containing GP2 protein (Tordai et al. (1999) FEBS Lett.461(1-2):63-67; Wong & Lowe, (1996) Gene, 171(2):311-312). GP2 plays animportant role in the secretion of pancreatic digestive enzymes. GP2 isthe major glycoprotein component of the zymogen granule membrane.Proteolytic processing of GP2 and its release from the zymogen granulemembrane occur as part of the normal process of zymogen granulesecretion in the pancreas (Fritz et al. (2002) Pancreas, 24(4):336-343).

Proteins such as PANZP1 and PANZP2 that are localized in the nematodegut are especially attractive targets for the development of vaccines.Although gut-localized proteins are accessible to antibodies, they arenormally inaccessible to host immune surveillance that is required tomount an immune response. Nevertheless, these so-called “hiddenantigens”, when purified, can be used to stimulate highly effectiveantibody responses in animals, especially against blood-feedingnematodes (Munn (1997) Int. J. Parasit. 27(4):359-366); Newton & Munn(1999) Parasitology Today, 15:116-122).

The structural features of the PANZP1 and PANZP2 suggest possiblestrategies for the production of antibodies and for the rational designof peptide inhibitors that could interfere with the protein-proteininteractions mediated by the PAN and ZP domain portions of the molecule.It has been shown in studies with blood coagulation factors thatantibodies and peptides that compete for binding to PAN domains disruptthe normal protein-protein interactions, and prevent blood coagulation(Baglia et al. (1995) J. Biol. Chem. 270(12):6734-6740; Sun & Gailani(1996) J. Biol. Chem. 271(46):29023-29028; Renne et al. (2002) J. Biol.Chem. 277(7):4892-4899). Recombinant proteins containing the PAN domainonly have been shown to assume the proper conformation, suggesting thatit would be possible to purify amounts of PAN domains that could be usedfor the production of antibodies (Baglia & Walsh (1996) J. Biol. Chem.271(7):3652-3658; Baglia et al. (2000) J. Biol. Chem275(41):31954-31962). It has also been demonstrated that syntheticpeptides that are designed from conformationally constrained portions ofthe PAN domain sequence (i.e., peptides which have at least one of theconserved disulfide linkages) are effective inhibitors of the normalprotein-protein interaction carried out by the whole protein. Nematoderesistant transgenic plants may be created by the production ofplantibodies capable of interfering with the function of PANZP1 orPANZP2 or the expression in plants of peptides or individual PAN or ZPdomains that can interfere with the normal functioning of nematodePANZP1 or PANZP2 in dominant negative fashion. The small size ofpeptides or individual domains may be an advantage for applicationsagainst certain plant parasitic nematodes, which appear to have sizeexclusion constraints for oral uptake.

The present invention provides nucleic acid sequences from nematodesencoding PAN and ZP containing receptor-like polypeptides. The S.stercoralis nucleic acid molecule (SEQ ID NO: 1) and the encoded PANZP1(SEQ ID NO: 3) are depicted in FIGS. 1A-1C. The S. stercoralis nucleicacid molecule (SEQ ID NO: 2) and the encoded PANZP2 (SEQ ID NO: 4) aredepicted in FIGS. 2A-2B. The M. javanica nucleic acid molecule (SEQ IDNO: 7) and the encoded PANZP1 (SEQ ID NO: 10) are depicted in FIGS.3A-3C. The partial H. glycines nucleic acid molecule (SEQ ID NO: 8) andthe encoded PANZP1 (SEQ ID NO: 11) are depicted in FIG. 4. The partialB. malayi nucleic acid molecule (SEQ ID NO: 9) and the encoded PANZP1(SEQ ID NO: 12) are depicted in FIGS. 5A-5B. Certain sequenceinformation for the PANZP1 and PANZP2 genes described herein issummarized in Table 1, below.

TABLE 1 Species CDNA ORF Polypeptide Figure S. stercoralis SEQ ID SEQ IDSEQ ID FIGS. 1A–1C NO: 1 NO: 5 NO: 3 S. stercoralis SEQ ID SEQ ID SEQ IDFIGS. 2A–2B NO: 2 NO: 6 NO: 4 M. javanica SEQ ID SEQ ID SEQ ID FIGS.3A–3C NO: 7 NO: 13 NO: 10 H. glycines SEQ ID SEQ ID SEQ ID FIG. 4 NO: 8NO: 14 NO: 11 B. malayi SEQ ID SEQ ID SEQ ID FIGS. 5A–5B NO: 9 NO: 15NO: 12

The invention is based, in part, on the discovery of PANZP sequencesfrom S. stercoralis, M. javanica, H. glycines, and B. malayi. Thefollowing examples are, therefore, to be construed as merelyillustrative, and not limitative of the remainder of the disclosure inany way whatsoever. All of the publications cited herein are herebyincorporated by reference in their entirety.

EXAMPLES

A TBLASTN query with the C. elegans genes C34G6.6

(gi|17505859|ref|NP_(—)491706.1|; PANZP1) and F52B11.3(gi|17540572|ref|NP_(—)502699.1|; PANZP2) identified multiple expressedsequence tags (ESTs are short nucleic acid fragment sequences fromsingle sequencing reads) in dbest that are predicted to encode a portionof PANZP enzymes in multiple nematode species.

PANZP ESTs identified as similar to C. elegans C34G6.6 (C. elegansPANZP1) include but are not limited to Brugia malayi(gi|2199168|gb|AA471404.1|AA471404); Pristionchus pacificus(gi|15339536|gb|BI500192.1|BI500192); Strongyloides stercoralis

(gi|9830619|gb|BE579677.1|BE579677); Ascaris summ

(gi|15785830|gb|BI782938.1|BI782938); Meloidogyne javanica

(gi|15766417|gb|BI744615.1|BI744615); Strongyloides ratti

(gi|14494496|gb|BI073876.1|BI073876); and Haemonchus contortus

(gi|10818965|gb|BF060055.1|BF060055).

PANZP ESTs identified as similar to C. elegans F52B11.3 (C. elegansPANZP2) include but are not limited to Strongyloides ratti(gi|14288440|gb|BG893830.1|BG893830); Strongyloides stercoralis(gi|9831122|gb|BE580180.1|BE580180); Meloidogyne hapla

(gi|19435833|gb|BM952243.1|BM952243); Brugia malayi

(gi|2605443|gb|AA661399.1|AA661399); and Onchocerca volvulus

(gi|14624150|gb|BI142440.1|BI142440).

Full-Length PAN and ZP Containing Receptor-Like cDNA Sequences

Plasmid clone, Div3206, corresponding to the S. stercoralis EST sequence( GENBANK® Identification No: 9831352) was obtained from the GenomeSequencing Center (St. Louis, MO). The cDNA insert in the plasmid wassequenced in its entirety. Unless otherwise indicated, all nucleotidesequences determined herein were sequenced with an automated DNAsequencer (such as model 373 from Applied Biosystems, Inc.) usingprocesses well-known to those skilled in the art. Primers used forsequencing are listed in Table 2 (see below). Partial sequence data forthe S. stercoralis PANZP1 was obtained from Div3206, includingnucleotide sequence for codons 141-1122 and additional 3′untranslatedsequence. To obtain the missing 5′-sequence of the S. stercoralis PANZP1gene, the 5′-oligo-capped RACE method (GENERACER™ kit from InvitrogenLife Technologies) was applied. This technique results in the selectiveligation of an RNA oligonucleotide (SEQ ID NO: 22) to the 5′-ends ofdecapped mRNA using T4 RNA ligase. First strand cDNA synthesis fromtotal S. stercoralis oligo-capped RNA was performed using an internalgene specific primer (PN1ss-5; SEQ ID NO: 23), designed from the knownsequence, that anneals within the cDNA molecule of interest. The firststrand cDNA was then directly PCR amplified using a nested gene specificprimer (PN1ss-2; SEQ ID NO: 24) designed from known sequence thatanneals within the cDNA molecule of interest, and the GENERACER™ 5′nested oligo (SEQ ID NO: 26), which is homologous to the 5′-end of allcDNAs amplified with the GENERACER™ oligo-capped RNA method. Thisprocedure was performed to generate clone Div3577, which contains codons1-184 in addition to 5′ untranslated sequences. Taken together, clonesDiv3206 and Div3577 contain sequences comprising the complete openreading frame of the PANZP1 gene of S. stercoralis.

Plasmid clone, Div3172, corresponding to the S. stercoralis EST sequence(GENBANK® Identification No: 9830179) was obtained from the GenomeSequencing Center (St. Louis, Mo.). The cDNA insert in the plasmid wassequenced in its entirety. Primers used for sequencing are listed inTable 2. Full sequence data for the S. stercoralis PANZP2 was obtainedfrom Div3 172, including nucleotide sequence for codons 1-557 andadditional 5′-and 3′-untranslated sequences. Div3172 contains thecomplete open reading frame of the PANZP2 gene of S. stercoralis.

Plasmid clone, Div2577, corresponding to the M javanica EST sequence(GENBANK® Identification No: 15766417) was obtained from the GenomeSequencing Center (St. Louis, Mo.). The cDNA insert in the plasmid wassequenced in its entirety. Partial sequence data for the M javanicaPANZP1 was obtained from Div2577, including nucleotide sequence forcodons 100-233. The available sequence lacked the first 99 codons andthe last 977 codons of the M javanica PANZP1, as well as 5′ and 3′untranslated regions. To obtain the middle region of the M javanicaPANZP1 gene, the 3′ RACE technique was applied. First strand cDNAsynthesis from total M javanica RNA was performed using an oligo dTprimer (SEQ ID NO: 21). The cDNA was then directly PCR amplified using agene specific primer (PAN18; SEQ ID NO: 37) designed from the knownsequence that anneals within the cDNA molecule of interest, and adegenerate primer (PAN25; SEQ ID NO: 38) designed to anneal to region ofthe gene predicted to exhibit strong homology shared across manynematode PANZP1 genes. This procedure was performed to generate cloneDiv3651, which contains codons 164-913.

To obtain the missing 5′ end of the M. javanica PANZP1 gene, the 5′ RACEtechnique was applied. First strand cDNA synthesis from total M.javanica RNA was performed using a gene specific primer (Mj-P1-R2; SEQID NO: 40). Single stranded cDNA was then dC-tailed and PCR amplifiedusing a gene specific primer (Mj-P1-R3; SEQ ID NO: 41) and the AAP(abridged anchor primer) (SEQ ID NO: 50) A final nested PCR wasperformed using gene specific primer Mj-P1-R4 (SEQ-ID NO: 42) and AUAP(abridged universal primer) (SEQ ID NO: 19). This procedure wasperformed to generate clone Div4453, which contains codons 1-118.

To obtain the 3′ end of the M. javanica PANZP 1 gene, the 3′ RACEtechnique was applied. First strand cDNA synthesis from M. javanica RNAwas performed as described previously. The first strand cDNA wasdirectly PCR amplified using a gene specific primer (P1-Mj-F2; SEQ IDNO: 39) designed from the known sequence that anneals within the firststrand cDNA molecule of interest, and the AUAP primer (SEQ ID NO: 19),which is homologous to the 3′ end of the cDNA of interest. Thisprocedure was performed to generate clone Div4470, which contains codons846-1210 in addition to 3′ untranslated sequences. Taken together,clones Div2577, Div3651, Div4453, and Div4470 contain sequencescomprising the complete open reading frame of the PANZP1 gene of M.javanica.

Partial-Length PAN and ZP Containing Receptor-Like cDNA Sequences

In an attempt to obtain the H. glycines PANZP1 gene, first strand cDNAderived from total H. glycines RNA by reverse transcription with theOligo dT primer, was directly PCR amplified, using a degenerate primer(P1-10FA; SEQ ID NO: 43) designed to anneal to a region of stronghomology shared across many nematode PANZP1 genes, and anotherdegenerate primer (P1-02R; SEQ ID NO: 44). This procedure was performedto obtain clone Div4504, which contains codons 1-252. The H. glycinesPANZP1 gene fragment within plasmid Div4504 is missing the 5′ and 3′coding sequences. The encoded codons are arbitrarily numbered startingwith number 1 for convenience. The codons contained in the H. glycinesPANZP1 gene fragment correspond to codons 112-364 of the C. elegansPANZP1 gene.

Partial sequence data for the B. malayi PANZP1 was obtained from a B.malayi EST (GENBANK® Identification No: 2199168), including nucleotidesequence for codons 207-363. The available sequence lacked the first 206codons and, approximately, the last 700 codons of the B. malayi PANZP1,as well as the 5′ and 3′ untranslated regions. Partial sequence data forthe B. malayi PANZP1 was also obtained from the B. malayi EST (GENBANK®Identification No: 5342885), including nucleotide sequence for codons256-386. The available sequence lacked the first 255 codons and,approximately, the last 680 codons of the B. malayi PANZP 1, as well asthe 5′ and 3′ untranslated regions.

To obtain the middle region of the B. malayi PANZP 1 gene, the 3′ RACEtechnique was applied. First strand cDNA synthesis from total B. malayiRNA was performed using an oligo dT primer (SEQ ID NO: 21). The cDNA wasthen directly PCR amplified using a gene specific primer (PNbm-3; SEQ IDNO: 36) designed from the known sequence that anneals within the cDNAmolecule of interest, and a degenerate primer (PAN20; SEQ ID NO: 35)designed to anneal to region of strong homology shared across manynematode PANZP1 genes. This procedure was performed to generate cloneDiv3410, which contains codons 162-340. To obtain the 5′ sequence of theB. malayi PANZP1 gene, the 5′-oligo-capped RACE method GENERACER™ kitfrom Invitrogen Life Technologies) was applied. This technique resultsin the selective ligation of an RNA oligonucleotide (SEQ ID NO: 22) tothe 5′-ends of decapped mRNA using T4 RNA ligase. First strand cDNAsynthesis from total B. malayi oligo-capped RNA was performed using aninternal gene specific primer (PNbm-GR; SEQ ID NO: 31), designed fromthe known sequence, that anneals within the cDNA molecule of interest.The first strand cDNA was then directly PCR amplified using a nestedgene specific primer (PN1bm-GR-nest; SEQ ID NO: 32) designed from knownsequence that anneals within the cDNA molecule of interest, and theGENERACER™ 5′ nested oligo (SEQ ID NO: 26), which is homologous to the5′-end of all cDNAs amplified with the GENERACER™ oligo-capped RNAmethod. This procedure was performed to generaate clone Div3663, whichcontains codons 1-232, in addition to 5′-untranslated sequences. Toobtain more of the 3′ sequence of the B. malayi PANZP1 gene, the 3′ RACEtechnique was applied. First strand cDNA synthesis from total B. malayiRNA was performed using an oligo dT primer (SEQ ID NO: 21). The cDNA wasthen directly PCR amplified using a gene specific primer (PNbm-5; SEQ IDNO: 33) designed from the known sequence that anneals within the cDNAmolecule of interest, and a degenerate primer (PAN23; SEQ ID NO: 34)designed to anneal to region of strong homology shared across manynematode PANZP 1 genes. This procedure was performed to generate cloneDiv3643, which contains codons 305-881. Taken together, clones Div3410,Div3663, and Div3643 contain sequences comprising approximately 75% ofthe complete B. malayi PANZP1 open reading frame. The 3′ end sequencehas yet to be completed.

TABLE 2 SEQ ID Name Sequence NO Homology to AUAP ggccacgcgtcgactagtac 19abridged universal primer (homolgous to the 5′ ends of primers Oligo dTand AAP) SL1 gggtttaattacccaagtttga 20 nematode transpliced leader OligodT ggccacgcgtcgactagtacttttttttttttttttt 21 universal primer to poly Atail RNA oligo cgacuggagcacgaggacacugacauggacugaaggaguagaaa 22GENERACER ™ RNA oligo PN1ss-5 ccgtccaagaggctttgaac 23 Ss PANZP1 (codons274–279) PN1ss-2 gatctggtcgatcaagtc 24 Ss PANZP1 (codons 180–184) GR5cgactggagcacgaggacactga 25 GENERACER ™ 5′ primer GR5nggacactgacatggactgaaggagta 26 GENERACER ™ 5′ nested primer AN07.C09tcagtgacgttatgtcctcc 27 Ce PANZP1 genomic AN07.D09 tgacagatggaacattctcc28 Ce PANZP1 genomic AN08.A10 acttcaggacacgacttgac 29 Ce PANZP2 genomicAN08.B10 caatcagagatggtaactcc 30 Ce PANZP2 genomic PNbm-GRcgttgtagacagtcgctgagtacata 31 Bm PANZP1 (codons 247–254) PN1bm-GR-nccaactcgttagctagctgacg 32 Bm PANZP1 (codons 226–232) PNbm-5cgaacatgtcgcaatgtac 33 Bm PANZP1 (codons 305–310) PAN23catngccatdatytccca 34 PANZP1 degenerate (codons 876–881) PAN20ttyggnttygartgygar 35 PANZP1 degenerate (codons 162–167) PNbm-3gatcgaggcacatcgttac 36 Bm PANZP1 (codons 335–340) PAN18gtttagatgctgttgatac 37 Mj PANZP1 (codons 164–168) PAN25tcdatyttnccyctnggytg 38 Mj PANZP1 degenerate (codons 908–913) P1-Mj-F2caagatatggacaatggaac 39 Mj PANZP1 (codons 846–851) Mj-P1-R2atacattcggcatccaatgg 40 Mj PANZP1 (codons 181–186) Mj-P1-R3actgactcgcattcaaagcc 41 Mj PANZP1 (codons 171–176) Mj-P1-R4tagctaatctagctagtgtc 42 Mj PANZP1 (codons 113–118) P1-10FAgarcaraaratgctngt 43 Hg PANZP1 (codons 1–6) P1-02R tgytcrttrtartartacat44 Hg PANZP1 (codons 247–252) T7 gtaatacgactcactatagggc 47 vectorpolylinker primer T3 aattaaccctcactaaaggg 48 vector polylinker primerSP6 gatttaggtgacactatag 49 vector polylinker primer AAPggccacgcgtcgactagtacggggggggg 50 abridged anchor primerCharacterization of Nematode PAN and ZP Containing Receptor-LikeProteins

The sequences of the two PANZP-like nucleic acid molecules (PANZP1 andPANZP2 from S. stercoralis, respectively) are depicted in FIGS. 1A-1Cand FIGS. 2A-2B as SEQ ID NO: 1 and SEQ ID NO: 2. The open reading frameof SEQ ID NO: 1 (SEQ ID NO: 5) contains an open reading frame encoding a1122 amino acid polypeptide (SEQ ID NO:3). The open reading frame of SEQID NO: 2 (SEQ ID NO: 6) contains an open reading frame encoding a 557amino acid polypeptide (SEQ ID NO: 4).

The S. stercoralis PANZP1 protein (FIGS. 1A-1C: SEQ ID NO: 3) isapproximately 54% identical (in the region of shared homology) to the C.elegans PANZP1 proteins (FIG. 4; SEQ ID NOs: 7 and 8). The similaritybetween the PANZP1 proteins from S. stercoralis and from C. elegans ispresented as a multiple alignment generated by the ClustalX multiplealignment program as described below (FIG. 7).

The S. stercoralis PANZP2 protein (FIGS. 2A-2B; SEQ ID NO: 4) isapproximately 79% identical (in the region of shared homology) to the C.elegans PANZP2 protein (FIGS. 5A-5B; SEQ ID NO: 9). The similaritybetween the PANZP2 proteins from S. stercoralis and from C. elegans ispresented as a multiple alignment generated by the ClustalX multiplealignment program as described below (FIG. 8).

The sequences of PANZP1-like nucleic acid molecules from M. javanica, H.glycines, and B. malayi are depicted in FIGS. 3A-3C, 4, and 5 as SEQ IDNO: 7, 8, and 9 respectively. The open reading frames within SEQ ID NO:7-9 are shown as SEQ ID NO: 13-15 respectively. The M. javanicaPANZP1-like sequence encodes a predicted polypeptide of 1210 amino acids(SEQ ID NO: 10). The partial H. glycines PANZP 1-like sequence encodes apredicted polypeptide of 250 amino acids (SEQ ID NO: 11). The partial B.malayi PANZP 1-like sequence encodes a predicted polypeptide of 881amino acids (SEQ ID NO: 12).

The M. javanica PANZP1 protein (FIGS. 3A-3C: SEQ ID NO: 10) isapproximately 46% identical (in the region of shared homology) to the C.elegans PANZP1 proteins (FIG. 7; SEQ ID NO: 17). The similarity betweenthe PANZP1 proteins from M. javanica and from C. elegans is presented asa multiple alignment generated by the ClustalX multiple alignmentprogram as described below (FIG. 7).

The H. glycines PANZP1 protein (FIG. 4: SEQ ID NO: 11) is approximately65% identical (in the region of shared homology) to the C. elegansPANZP1 proteins (FIG. 7; SEQ ID NO: 17). The similarity between thePANZP1 proteins from H. glycines and from C. elegans is presented as amultiple alignment generated by the ClustalX multiple aligmnent programas described below (FIG. 7).

The B. malayi PANZP1 protein (FIGS. 5A-5B: SEQ ID NO: 12) isapproximately 56% identical (in the region of shared homology) to the C.elegans PANZP1 proteins (FIG. 7; SEQ ID NO: 17). The similarity betweenthe PANZP1 proteins from B. malayi and from C. elegans is presented as amultiple alignment generated by the ClustalX multiple alignment programas described below (FIG. 7).

Hidden Markov Model based domain analysis of the nematode PAN and ZPcontaining receptor-like proteins using the PFAM database (available onthe internet at pfam.wustl.edu) shows that the nematode PANZP1 proteinscontain six PAN domains and a single ZP domain. Different PANZP proteinshave different numbers of PAN domains (e.g., C. elegans PANZP2 has fourPAN domains) but the overall module arrangement is the same (i.e.,secretion leader, (PAN)_(x), ZP, T_(M)). In PANZP1 the seven domains arereferred to as PAN1, PAN2, PAN3, PAN4, PAN5, PAN6 and ZP. The predictedamino acid positions of these domains in the PANZP proteins are listedin the table below.

TABLE 3 Amino Acid positions of conserved PAN and ZP motifs in NematodePANZP proteins Nematode PAN1 PAN2 PAN3 PAN4 PAN5 PAN6 ZP S. stercoralis32–108 115–201 206–295 302–392 399–485 508–580 708–999 PANZP1 (SEQ IDNO: 3) M. javanica 36–122 129–215 220–309 316–406 413–499 520–603 731–1045 PANZP1 (SEQ ID NO: 10) H. glycines  1–77 106–196 198–250PANZP1 (SEQ ID NO: 11) B. malayi 40–114 121–207 212–300 307–397 404–490497–575 PANZP1 (SEQ ID NO: 12) C. elegans _(a) 25–97  104–190 212–300307–397 404–491 504–576 652–953 PANZP1 C. elegans _(b) 25–97  104–190212–300 307–397 404–491 508–580 656–957 PANZP1 C. briggsae 25–97 104–190 211–299 306–396 403–490 507–579 655–956 PANZP1 S. stercoralis23–114 124–207 215–298 308–385 384–442 PANZP2 (SEQ ID NO: 4) C. elegans21–112 122–204 212–295 305–382 391–632 PANZP2 C. briggsae 22–113 123–205213–296 306–383 392–633 PANZP2

The similarity between S. stercoralis, M. javanica, H. glycines, and B.malayi PANZP sequences and other sequences was also investigated bycomparison to sequence databases using BLASTP analysis against nr (anon-redundant protein sequence database available on the internet atncbi.nlm.nih.gov) and TBLASTN analysis against dbest (an EST sequencedatabase available on the internet at ncbi.nlm.nih.gov; top 500 hits;E=1e−4). The “Expect (E) value” is the number of sequences that arepredicted to align by chance to the query sequence with a score S orgreater given the size of the database queried. This analysis was usedto determine the potential number of plant and vertebrate homologs foreach of the nematode PANZP polypeptides described above. None of thePANZP sequences described above had high scoring vertebrate hits in nror dbest having sufficient sequence similarity to meet the threshold Evalue of 1 e−4 (this E value approximately corresponds to a thresholdfor removing sequences having a sequence identity of less than about 25%over approximately 100 amino acids). Accordingly, the PANZP enzymes ofthis invention do not appear to share significant sequence similaritywith common vertebrate PAN containing proteins such as the Homo sapiensPlasminogen (gi|4505881|ref|NP_(—)000292.1|) or ZP containing proteinssuch as the Homo sapiens Zona Pellucida 2 glycoprotein(gi|4508045|ref|NP_(—)003451.1|).

On the basis of the lack of similarity to vertebrate PAN or ZPcontaining proteins and the lack of significant plant homologs, thePANZP enzymes are useful targets of inhibitory (small molecule, peptideor protein) compounds selective for nematodes over their hosts (e.g.,humans, animals, and plants).

Functional predictions were made using BLAST with the default parameterson the nr database. BLAST searches and multiple alignment constructionwith CLUSTALX demonstrated that the C. elegans genes C34G6.6a, C34G6.6band F52B11.3 define a family of PAN and ZP containing proteins found innematodes and arthropods (e.g., Anopheles gambiae, C. briggsae andDrosophila melanogaster). Reciprocal blast searches and phylogenetictrees confirm that the nucleotide sequences from S. stercoralis, M.javanica, H. glycines, and B. malayi are orthologs of the C. elegans andC. briggsae genes and are therefore members of the same PANZP family ofproteins. Protein localizations were predicted using the TargetP server(available on the internet at cbs.dtu.dk/services/TargetP) andtransmembrane domains with the TMHMM server (available on the internetat cbs.dtu.dk/services/TMHMM). The nematode PANZP polypeptides (SEQ IDNO: 3.4, 10, 11, and 12), like the C. elegans and C. briggsae proteins(SEQ ID NO: 7, 8, 9, 45 and 46), are likely extracellular transmembraneproteins because of the presence of strong secretion leaders, C-terminaltransmembrane domains and PAN and ZP domains that are likelyglycosylated. Additionally, some fraction of the PANZP proteins may becleaved from the membrane (e.g., at a polybasic site after the ZPdomain) by the action of an endoproteinases (e.g., a furin-typeendopeptidase).

RNA Mediated Interference (RNAi)

A double stranded RNA (dsRNA) molecule can be used to inactivate a geneencoding a PAN and ZP domain protein (PANZP) in a cell by a processknown as RNA mediated-interference (Fire et al. (1998) Nature391:806-811, and Gönczy et al. (2000) Nature 408:331-336). The dsRNAmolecule can have the nucleotide sequence of a PANZP nucleic acid(preferably exonic) or a fragment thereof. For example, the molecule cancomprise at least 50, at least 100, at least 200, at least 300, or atleast 500 or more contiguous nucleotides of a PANZP gene. The dsRNAmolecule can be delivered to nematodes via direct injection, by soakingnematodes in aqueous solution containing concentrated dsRNA, or byraising bacteriovorous nematodes on E. coli genetically engineered toproduce the dsRNA molecule (Kamath et al. (2000) Genome Biol. 2; Tabaraet al. (1998) Science 282:430-431).

PANZP RNAi by Feeding

C. elegans were grown on lawns of E. ccli genetically engineered toproduce double-stranded RNA (dsRNA) designed to inhibit PANZP1 or PANZP2expression in order to investigate whether PANZP1 or PANZP2 expressionis essential. Briefly, E. coli were transformed with genomic fragmentsencoding portions of the C. elegans PANZP1 or the PANZP2 gene. A 1048nucleotide fragment was amplified from the PANZP1 gene usingoligo-nucleotide primers containing the sequences 5′-TCAGTGACGTTATGTCCTCC-3′ (SEQ ID NO: 51) and 5′-TGACAGATGGAACATTCTCC-3′(SEQ ID NO: 52). A 926 nucleotide fragment was amplified from the PANZP2gene using oligo-nucleotide primers containing the sequences5′-ACTTCAGGACACGACTTGAC-3′ (SEQ lID NO: 53) and5′-CAATCAGAGATGGTAACTCC-3′ (SEQ ID NO:54) respectively. The clonedPANZP1 and PANZP2 genomic fragments were cloned separately into an E.coli expression vector between opposing T7 polymerase promoters. Theexpression clones were separately transformed into a strain of E. colithat carries an IPTG-inducible T7 polymerase. As a control, E. coli wastransformed with a gene encoding the Green Fluorescent Protein (GFP).

Feeding RNAi was initiated from C. elegans L4 larvae at 23° C. on NGMplates containing IPTG and E. coli expressing the C. elegans PANZP1dsRNA, PANZP2 dsRNA or GFP dsRNA. C. elegans exposed to E. coliexpressing PANZP1 dsRNA or PANZP2 dsRNA exhibited severe reduction inbrood size of the fed or P0 animal. In addition, of the eggs laid, onlya fraction hatched, and the hatched animals died at the L1 or L2 larvalstage. The sequence of the PANZP1 and PANZP2 genes is of sufficientlyhigh complexity (i.e., unique) such that the RNAi is not likely torepresent cross reactivity with other genes.

C. elegans cultures grown in the presence of E. coli expressing dsRNAfrom the PANZP1 or the PANZP2 gene were strongly impaired indicatingthat the PANZP genes provide essential functions in nematodes and thatdsRNA from the PAN and ZP containing receptor-like genes is lethal wheningested by C. elegans. These results demonstrate that PANZPs areimportant for the viability of C. elegans and suggest that they areuseful targets for the development of compounds (small molecule,peptide, protein or otherwise) that reduce the viability of nematodes.

Orthologs of PANZP1 are Present in Intestinal cDNA Libraries

An expressed sequence tag (EST) apparently encoding an orthologue ofPANZP1 was identified from an Ascaris suum intestinal cDNA. The presenceof a PANZP1 orthologue in an intestinal library suggests PANZP1 isexpressed in the nematode intestine. In addition, the PANZP1 proteinsequence contains sequences suggesting that PANZP1 is a transmembraneprotein and that the PAN domains are extracellular. Together, theseobservations indicate that the PAN domains of PANZP1 may be accessibleto drugs, peptides or proteins (e.g., antibodies) ingested by the worm.

PAN domains have been shown to be involved in protein-proteininteractions in other systems (Renne et al. (2002) J. Biol. Chem.277(7):4892-9). Therefore, one approach to inactivating the function ofPANZP polypeptides is to interfere with protein-protein interactionsusing an antibody against a PAN domain, a peptide comprising a PANdomain or a portion of a PAN domain, or any peptide capable of strongintereaction with a native PAN domain. These entities may act asdominant negatives that will block the function of PANZP1 proteins. Theintact protein fragments thereof can, for example, be over-expressed inplants where they could negatively interact with PANZP proteins of plantparasitic nematodes upon ingestion by the nematodes. Alternatively theintact proteins or fragments could be injected into or fed to a hostanimal and thus disrupt the function of animal parasitic nematode PANZPproteins upon ingestion by the nematodes. Since PANZP1 performs anessential function, entities that disrupt its function will haveanthelmintic properties.

Identification of Additional PAN and ZP Domain Containing Receptor-LikeSequences

A skilled artisan can utilize the methods provided in the example aboveto identify additional nematode PAN and ZP domain containingreceptor-like sequences, e.g., PANZP sequences from nematodes other thanS. stercoralis, M. javanica, H. glycines, B. malayi, or C. elegans. Inaddition, nematode PANZP sequences can be identified by a variety ofmethods including computer-based database searches, hybridization-basedmethods, and functional complementation.

Database Identification

A nematode PAN and ZP containing receptor-like sequence can beidentified from a sequence database, e.g., a protein or nucleic aciddatabase using a sequence disclosed herein as a query. Sequencecomparison programs can be used to compare and analyze the nucleotide oramino acid sequences. One such software package is the BLAST suite ofprograms from the National Center for Biotechnology Institute (NCBI;Altschul et al. (1997) Nucl. Acids Research 25:3389-3402). A PAN and ZPcontaining receptor-like sequence of the invention can be used to querya sequence database, such as nr, dbest (expressed sequence tag (EST)sequences), and htgs (high-throughput genome sequences), using acomputer-based search, e.g., FASTA, BLAST, or PSI-BLAST search.Homologous sequences in other species (e.g., plants and animals) can bedetected in a PSI-BLAST search of a database such as nr (E value=10, Hvalue=1e-2, using, for example, four iterations; available atwww.ncbi.nlm.nih.gov). Sequences so obtained can be used to construct amultiple alignment, e.g., a ClustalX alignment, and/or to build aphylogenetic tree, e.g., in ClustalX using the Neighbor-Joining method(Saitou et al. (1987) Mol. Biol. Evol. 4:406-425) and bootstrapping(1000 replicates; Felsenstein (1985) Evolution 39:783-791). Distancesmay be corrected for the occurence of multiple substitutions[D_(corr)=−1n(1−D−D²/5) where D is the fraction of amino aciddifferences between two sequences] (Kimura (1983) The Neutral Theory ofMolecular Evolution, Cambridge University Press).

The aforementioned search strategy can be used to identify PAN and ZPdomain containing receptor-like sequences in nematodes of the followingnon-limiting, exemplary genera: Plant-parasitic nematode genera: Afrina,Anguina, Aphelenchoides, Belonolaimus, Bursaphelenchus, Cacopaurus,Cactodera, Criconema, Criconemoides, Cryphodera, Ditylenchus,Dolichodorus, Dorylaimus, Globodera, Helicotylenchus, Hemicriconemoides,Hemicycliophora, Heterodera, Hirschmanniella, Hoplolaimus, Hypsoperine,Longidorus, Meloidogyne, Mesoanguina, Nacobbus, Nacobbodera,Panagrellus, Paratrichodorus, Paratylenchus, Pratylenchus,Pterotylenchus, Punctodera, Radopholus, Rhadinaphelenchus,Rotylenchulus, Rotylenchus, Scutellonema, Subanguina, Thecavermiculatus,Trichodorus, Turbatrix, Tylenchorhynchus, Tylenchulus, Xiphinema.

Animal- and human-parasitic nematode genera: Acanthocheilonema,Aelurostrongylus, Ancylostoma, Angiostrongylus, Anisakis, Ascaris,Ascarops, Bunostomum, Brugia, Capillaria, Chabertia, Cooperia,Crenosoma, Cyathostome species (Small Strongyles), Dictyocaulus,Dioctophyma, Dipetalonema, Dirofiliaria, Dracunculus, Draschia,Elaneophora, Enterobius, Filaroides, Gnathostoma, Gonylonema, Habronema,Haemonchus, Hyostrongylus, Lagochilascaris, Litomosoides, Loa,Mammomonogamus, Mansonella, Muellerius, Metastrongylid, Necator,Nematodirus, Nippostrongylus, Oesophagostomum, Ollulanus, Onchocerca,Ostertagia, Oxyspirura, Oxyuris, Parafilaria, Parascaris,Parastrongyloides, Parelaphostrongylus, Physaloptera, Physocephalus,Protostrongylus, Pseudoterranova, Setaria, Spirocerca, Stephanurus,Stephanofilaria, Strongyloides, Strongylus, Spirocerca, Syngamus,Teladorsagia, Thelazia, Toxascaris, Toxocara, Trichinella,Trichostrongylus, Trichuris, Uncinaria, and Wuchereria.

Particularly preferred nematode genera include: Plant: Anguina,Aphelenchoides, Belonolaimus, Bursaphelenchus, Ditylenchus,Dolichodorus, Globodera, Heterodera, Hoplolaimus, Longidorus,Meloidogyne, Nacobbus, Pratylenchus, Radopholus, Rotylenchus,Tylenchulus, Xiphinema.

Animal and human: Ancylostoma, Ascaris, Brugia, Capillaria, Cooperia,Cyathostome species, Dictyocaulus, Dirofiliaria, Dracunculus,Enterobius, Haemonchus, Necator, Nematodirus, Oesophagostomum,Onchocerca, Ostertagia, Oxyspirura, Oxyuris, Parascaris, Strongyloides,Strongylus, Syngamus, Teladorsagia, Thelazia, Toxocara, Trichinella,Trichostrongylus, Trichuris, and Wuchereria.

Particularly preferred nematode species include: Plant: Anguina tritici,Aphelenchoides fragariae, Belonolaimus longicaudatus, Bursaphelenchusxylophilus, Ditylenchus destructor, Ditylenchus dipsaci Dolichodorusheterocephalous, Globodera pallida, Globodera rostochiensis, Globoderatabacum, Heterodera avenae, Heterodera cardiolata, Heterodera carotae,Heterodera cruciferae, Heterodera glycines, Heterodera major, Heteroderaschachtii, Heterodera zeae, Hoplolaimus tylenchiformis, Longidorussylphus, Meloidogyne acronea, Meloidogyne arenaria, Meloidogynechitwoodi, Meloidogyne exigua, Meloidogyne graminicola, Meloidogynehapla, Meloidogyne incognita, Meloidogyne javanica, Meloidogyne nassi,Nacobbus batatiformis, Pratylenchus brachyurus, Pratylenchus coffeae,Pratylenchus penetrans, Pratylenchus scribneri, Pratylenchus zeae,Radopholus similis, Rotylenchus reniformis, Tylenchulus semipenetrans,Xiphinema americanum.

Animal and human: Ancylostoma braziliense, Ancylostoma caninum,Ancylostoma ceylanicum, Ancylostoma duodenale, Ancylostoma tubaeforme,Ascaris suum, Ascaris lumbrichoides, Brugia malayi, Capillaria bovis,Capillaria plica, Capillaria feliscati, Cooperia oncophora, Cooperiapunctata, Cyathostome species, Dictyocaulus filaria, Dictyocaulusviviparus, Dictyocaulus arnfieldi, Dirofiliaria immitis, Dracunculusinsignis, Enterobius vermicularis, Haemonchus contortus, Haemonchusplacei, Necator americanus, Nematodirus helvetianus, Oesophagostomumradiatum, Onchocerca volvulus, Onchocerca cervicalis, Ostertagiaostertagi, Ostertagia circumcincta, Oxyuris equi, Parascaris equorum,Strongyloides stercoralis, Strongylus vulgaris, Strongylus edentatus,Syngamus trachea, Teladorsagia circumcincta, Toxocara cati, Trichinellaspiralis, Trichostrongylus axei, Trichostrongylus colubriformis,Trichuris vulpis, Trichuris suis, Trichurs trichiura, and Wuchereriabancrofti.

Further, a PAN and ZP domain containing receptor-like sequence can beused to identify additional PANZP sequence homologs within a genome.Multiple homologous copies of a PANZP sequence can be present. Forexample, a nematode PANZP sequence can be used as a seed sequence in aniterative PSI-BLAST search (default parameters, substitutionmatrix=Blosum62, gap open=11, gap extend=1) of a non redundant databasesuch as wormpep (E value=1e−2, H value=1e−4, using, for example 4iterations) to determine the number of homologs in a database, e.g., ina database containing the complete genome of an organism. A nematodePANZP sequence can be present in a genome along with 1, 2, 3, 4, 5, 6,8, 10, or more homologs.

Hybridization Methods

A nematode PAN and ZP domain containing receptor-like sequence can beidentified by a hybridization-based method using a sequence providedherein as a probe. For example, a library of nematode genomic or cDNAclones can be hybridized under low stringency conditions with the probenucleic acid. Stringency conditions can be modulated to reducebackground signal and increase signal from potential positives. Clonesso identified can be sequenced to verify that they encode PANZPsequences.

Another hybridization-based method utilizes an amplification reaction(e.g., the polymerase chain reaction (PCR)). Oligonucleotides, e.g.,degenerate oligonucleotides, are designed to hybridize to a conservedregion of a PANZP sequence (e.g., a region conserved in the nematodesequences depicted in FIGS. 7 and 8). The oligonucleotides are used asprimers to amplify a PANZP sequence from template nucleic acid from anematode, e.g., a nematode other than S. stercoralis, M. javanica, H.glycines, B. malayi, or C. elegans. The amplified fragment can be clonedand/or sequenced.

Full-Length cDNA and Sequencing Methods

The following methods can be used, e.g., alone or in combination withanother method described herein, to obtain full-length nematode PANZPgenes and determine their sequences.

Plant parasitic nematodes are maintained on greenhouse pot culturesdepending on nematode preference. Root Knot Nematodes (Meloidogyne sp)are propagated on Rutgers tomato (Burpee), while Soybean Cyst Nematodes(Heterodera sp) are propagated on soybean. Total nematode RNA isisolated using the TRIZOL reagent (Gibco BRL). Briefly, 2 ml of packedworms are combined with 8 ml TRIZOL reagent and solubilized byvortexing. Following 5 minutes of incubation at room temperature, thesamples are divided into smaller volumes and spun at 14,000×g for 10minutes at 4° C. to remove insoluble material. The liquid phase isextracted with 200 μl of chloroform, and the upper aqueous phase isremoved to a fresh tube. The RNA is precipitated by the addition of 500μl of isopropanol and centrifuged to pellet. The aqueous phase iscarefully removed, and the pellet is washed in 75% ethanol and spun tore-collect the RNA pellet. The supernatant is carefully removed, and thepellet is air dried for 10 minutes. The RNA pellet is resuspended in 50μl of DEPC—H₂O and analyzed by spectrophotometry at λ 260 and 280 nm todetermine yield and purity. Yields can be 1-4 mg of total RNA from 2 mlof packed worms.

Full-length cDNAs can be generated using 5′ and 3′ RACE techniques incombination with EST sequence information. The molecular technique 5′RACE (Life Technologies, Inc., Rockville, Md.) can be employed to obtaincomplete or near-complete 5′ ends of cDNA sequences for nematode PANZPcDNA sequences. Briefly, following the instructions provided by LifeTechnologies, first strand cDNA is synthesized from total nematode RNAusing Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) and a genespecific “antisense” primer, e.g., designed from available EST sequence.RNase H is used to degrade the original mRNA template. The first strandcDNA is separated from unincorporated dNTPs, primers, and proteins usinga GlassMAX Spin Cartridge. Terminal deoxynucleotidyl transferase (TdT)is used to generate a homopolymeric dC tailed extension by thesequential addition of dCTP nucleotides to the 3′ end of the firststrand cDNA. Following addition of the dC homopolymeric extension, thefirst strand cDNA is directly amplified without further purificationusing Taq DNA polymerase, a gene specific “antisense” primer designedfrom available EST sequences to anneal to a site located within thefirst strand cDNA molecule, and a deoxyinosine-containing primer thatanneals to the homopolymeric dC tailed region of the cDNA in apolymerase chain reaction (PCR). 5′ RACE PCR amplification products arecloned into a suitable vector for further analysis and sequencing.

The molecular technique, 3′ RACE (Life Technologies, Inc., Rockville,Md.), can be employed to obtain complete or near-complete 3′ ends ofcDNA sequences for nematode PANZP cDNA sequences. Briefly, following theinstructions provided by Life Technologies (Rockville, Md.), firststrand cDNA synthesis is performed on total nematode RNA usingSuperScript™ Reverse Transcriptase and an oligo-dT primer that annealsto the polyA tail. Following degradation of the original mRNA templatewith RNase H, the first strand cDNA is directly PCR amplified withoutfurther purification using Taq DNA polymerase, a gene specific primerdesigned from available EST sequences to anneal to a site located withinthe first strand cDNA molecule, and a “universal” primer which containssequence identity to 5′ end of the oligo-dT primer. 3′ RACE PCRamplification products are cloned into a suitable vector for furtheranalysis and sequencing.

Nucleic Acid Variants

Isolated nucleic acid molecules of the present invention include nucleicacid molecules that have an open reading frame encoding a PANZPpolypeptide. Such nucleic acid molecules include molecules having: thesequences recited in SEQ ID NO: 1, 2, 7, 8, and 9 and the sequencecoding for the PANZP proteins recited in SEQ ID NO: 3, 4, 10, 11, and12. These nucleic acid molecules can be used, for example, in ahybridization assay to detect the presence of a S. stercoralis, M.javanica, H. glycines, or B. malayi nucleic acid in a sample.

The present invention includes nucleic acid molecules such as the onesshown in SEQ ID NO: 1, 2, 7, 8, and 9 that may be subjected tomutagenesis to produce single or multiple nucleotide substitutions,deletions, or insertions. Nucleotide insertional derivatives of thenematode gene of the present invention include 5′ and 3′ terminalfusions as well as intra-sequence insertions of single or multiplenucleotides. Insertional nucleotide sequence variants are those in whichone or more nucleotides are introduced into a predetermined site in thenucleotide sequence, although random insertion is also possible withsuitable screening of the resulting product. Deletion variants arecharacterized by the removal of one or more nucleotides from thesequence. Nucleotide substitution variants are those in which at leastone nucleotide in the sequence has been removed and a differentnucleotide inserted in its place. Such a substitution may be silent(e.g., synonymous), meaning that the substitution does not alter theamino acid defined by the codon. Alternatively, substitutions aredesigned to alter one amino acid for another amino acid (e.g.,non-synonymous). A non-synonymous substitution can be conservative ornon-conservative. A substitution can be such that activity, e.g., aPANZP activity, is not impaired. A conservative amino acid substitutionresults in the alteration of an amino acid for a similar acting aminoacid, or amino acid of like charge, polarity, or hydrophobicity, e.g.,an amino acid substitution listed in Table 4 below. At some positions,even conservative amino acid substitutions can disrupt the activity ofthe polypeptide.

TABLE 4 Conservative Amino Acid Replacements Amino acid Code Replacewith any of Alanine Ala Gly, Cys, Ser Arginine Arg Lys, His AsparagineAsn Asp, Glu, Gln, Aspartic Acid Asp Asn, Glu, Gln Cysteine Cys Met,Thr, Ser Glutamine Gln Asn, Glu, Asp Glutamic Acid Glu Asp, Asn, GlnGlycine Gly Ala Histidine His Lys, Arg Isoleucine Ile Val, Leu, MetLeucine Leu Val, Ile, Met Lysine Lys Arg, His Methionine Met Ile, Leu,Val Phenylalanine Phe Tyr, His, Trp Proline Pro Serine Ser Thr, Cys, AlaThreonine Thr Ser, Met, Val Tryptophan Trp Phe, Tyr Tyrosine Tyr Phe,His Valine Val Leu, Ile, Met

The current invention also embodies splice variants of nematode PANZPsequences.

Another aspect of the present invention embodies a polypeptide-encodingnucleic acid molecule that is capable of hybridizing under conditions oflow stringency (or high stringency) to the nucleic acid molecule putforth in SEQ ID NO: 1, 2, 7, 8, and 9 or their complements.

The nucleic acid molecules that encode for PAN and ZP domain containingreceptor-like polypeptides may correspond to the naturally occurringnucleic acid molecules or may differ by one or more nucleotidesubstitutions, deletions, and/or additions. Thus, the present inventionextends to genes and any functional mutants, derivatives, parts,fragments, naturally occurring polymorphisms, homologs or analogsthereof or non-functional molecules. Such nucleic acid molecules can beused to detect polymorphisms of PANZP genes, e.g., in other nematodes.As mentioned below, such molecules are useful as genetic probes; primersequences in the enzymatic or chemical synthesis of the gene; or in thegeneration of immunologically interactive recombinant molecules. Usingthe information provided herein, such as the nucleotide sequence SEQ IDNO: 1, 2, 7, 8, and 9, a nucleic acid molecule encoding an PANZPmolecule may be obtained using standard cloning and a screeningtechniques, such as a method described herein.

Nucleic acid molecules of the present invention can be in the form ofRNA, such as mRNA, or in the form of DNA, including, for example, cDNAand genomic DNA obtained by cloning or produced synthetically. The DNAmay be double-stranded or single-stranded. The nucleic acids may be inthe form of RNA/DNA hybrids. Single-stranded DNA or RNA can be thecoding strand, also referred to as the sense strand, or the non-codingstrand, also known as the anti-sense strand.

One embodiment of the present invention includes a recombinant nucleicacid molecule, which includes the isolated nucleic acid moleculesdepicted in SEQ ID NO: 1, 2, 7, 8, and 9, inserted in a vector capableof delivering and maintaining the nucleic acid molecule into a cell. TheDNA molecule may be inserted into an autonomously replicating vector(suitable vectors include, for example, pGEM3Z and pcDNA3, andderivatives thereof). The vector nucleic acid may be a bacteriophage DNAsuch as bacteriophage lambda or M13 and derivatives thereof. The vectormay be either RNA or DNA, single- or double-stranded, prokaryotic,eukaryotic, or viral. Vectors can include transposons, viral vectors,episomes, (e.g., plasmids), chromosomes inserts, and artificialchromosomes (e.g. BACs or YACs). Construction of a vector containing anucleic acid described herein can be followed by transformation of ahost cell such as a bacterium. Suitable bacterial hosts include, but arenot limited to, E. coli. Suitable eukaryotic hosts include yeast such asS. cerevisiae, other fungi, vertebrate cells, invertebrate cells (e.g.,insect cells), plant cells, human cells, human tissue cells, and wholeeukaryotic organisms. (e.g., a transgenic plant or a transgenic animal).Further, the vector nucleic acid can be used to generate a virus such asvaccinia or baculovirus.

The present invention also extends to genetic constructs designed forpolypeptide expression. Generally, the genetic construct also includes,in addition to the encoding nucleic acid molecule, elements that allowexpression, such as a promoter and regulatory sequences. The expressionvectors may contain transcriptional control sequences that controltranscriptional initiation, such as promoter, enhancer, operator, andrepressor sequences. A variety of transcriptional control sequences arewell known to those in the art and may be functional in, but are notlimited to, a bacterium, yeast, plant, or animal cell. The expressionvector can also include a translation regulatory sequence (e.g., anuntranslated 5′ sequence, an untranslated 3′ sequence, a poly A additionsite, or an internal ribosome entry site), a splicing sequence orsplicing regulatory sequence, and a transcription termination sequence.The vector can be capable of autonomous replication or it can integrateinto host DNA.

In an alternative embodiment, the DNA molecule is fused to a reportergene such as β-glucuronidase gene, β-galactosidase (lacZ),chloramphenicol-acetyltransferase gene, a gene encoding greenfluorescent protein (and variants thereof), or red fluorescent proteinfirefly luciferase gene, among others. The DNA molecule can also befused to a nucleic acid encoding a polypeptide affinity tag, e.g.glutathione S-transferase (GST), maltose E binding protein, protein A,FLAG tag, hexa-histidine, or the influenza HA tag. The affinity tag orreporter fusion joins the reading frames of SEQ ID NO: 1, 2, 7, 8,and/or 9 to the reading frame of the reporter gene encoding the affinitytag such that a translational fusion is generated. Expression of thefusion gene results in translation of a single polypeptide that includesboth a nematode PANZP region and reporter protein or affinity tag. Thefusion can also join a fragment of the reading frame of SEQ ID NO: 1, 2,7, 8, and/or 9. The fragment can encode a functional region of the PANZPpolypeptides, a structurally intact domain, or an epitope (e.g., apeptide of about 8, 10, 20, or 30 or more amino acids). A nematode PANZPnucleic acid that includes at least one of a regulatory region (e.g., a5′-regulatory region, a promoter, an enhancer, a 5′-untranslated region,a translational start site, a 3′-untranslated region, a polyadenylationsite, or a 3′-regulatory region) can also be fused to a heterologousnucleic acid. For example, the promoter of a PANZP nucleic acid can befused to a heterologous nucleic acid, e.g., a nucleic acid encoding areporter protein.

Suitable cells to transform include any cell that can be transformedwith a nucleic acid molecule of the present invention. A transformedcell of the present invention is also herein referred to as arecombinant or transgenic cell. Suitable cells can either be.untransformed cells or cells that have already been transformed with atleast one nucleic acid molecule. Suitable cells for transformationaccording to the present invention can either be: (i) endogenouslycapable of expressing the PANZP protein or; (ii) capable of producingsuch protein after transformation with at least one nucleic acidmolecule of the present invention.

In an exemplary embodiment, a nucleic acid of the invention is used togenerate a transgenic nematode strain, e.g., a transgenic C. elegansstrain. To generate such a strain, nucleic acid is injected into thegonad of a nematode, thus generating a heritable extrachromosomal arraycontaining the nucleic acid (see, e.g., Mello et al. (1991) EMBO J.10:3959-3970). The transgenic nematode can be propagated to generate astrain harboring the transgene. Nematodes of the strain can be used inscreens to identify inhibitors specific for a S. stercoralis, M.javanica, H. glycines, or B. malayi PANZP polypeptide.

Oligonucleotides

Also provided are oligonucleotides that can form stable hybrids with anucleic acid molecule of the present invention. The oligonucleotides canbe about 10 to 200 nucleotides, about 15 to 120 nucleotides, or about 17to 80 nucleotides in length, e.g., about 10, 20, 30, 40, 50, 60, 80,100, 120 nucleotides in length. The oligonucleotides can be used asprobes to identify nucleic acid molecules, primers to produce nucleicacid molecules, or therapeutic reagents to inhibit nematode PANZPprotein activity or production (e.g., antisense, triplex formation,ribozyme, and/or RNA drug-based reagents). The present inventionincludes oligonucleotides of RNA (ssRNA and dsRNA), DNA, or derivativesof either. The invention extends to the use of such oligonucleotides toprotect non-nematode organisms (for example e.g., plants and animals)from disease by reading the viability of infecting nematodes, e.g.,using a technology described herein. Appropriateoligonucleotide-containing therapeutic compositions can be administeredto a non-nematode organism using techniques known to those skilled inthe art, including, but not limited to, transgenic expression in plantsor animals.

Primer sequences can be used to amplify a PAN and ZP domain containingreceptor-like nucleic acid or fragment thereof. For example, at least 10cycles of PCR amplification can be used to obtain such an amplifiednucleic acid. Primers can be at least about 8-40, 10-30 or 14-25nucleotides in length, and can anneal to a nucleic acid “templatemolecule”, e.g., a template molecule encoding an PANZP genetic sequence,or a functional part thereof, or its complementary sequence. The nucleicacid primer molecule can be any nucleotide sequence of at least 10nucleotides in length derived from, or contained within sequencesdepicted in SEQ ID NO:1, 2, 7, 8, and/or 9 and their complements. Thenucleic acid template molecule may be in a recombinant form, in a virusparticle, bacteriophage particle, yeast cell, animal cell, plant cell,fungal cell, or bacterial cell. A primer can be chemically synthesizedby routine methods.

This invention embodies any PAN and ZP domain containing receptor-likesequences that are used to identify and isolate similar genes from otherorganisms, including nematodes, prokaryotic organisms, and othereukaryotic organisms, such as other animals and/or plants.

In another embodiment, the invention provides oligonucleotides that arespecific for a S. stercoralis, M. javanica, H. glycines, and B. malayiPANZP nucleic acid molecule. Such oligonucleotides can be used in a PCRtest to determine if a S. stercoralis, M. javanica, H. glycines, and/orB. malayi derived nucleic acid is present in a sample, e.g., to monitora disease caused S. stercoralis, M. javanica, H. glycines, and/or B.malayi.

Protein Production

Isolated PAN and ZP domain containing receptor-like proteins fromnematodes can be produced in a number of ways, including production andrecovery of the recombinant proteins and/or chemical synthesis of theprotein. In one embodiment, an isolated nematode PANZP protein isproduced by culturing a cell, e.g., a bacterial, fungal, plant, oranimal cell, capable of expressing the protein, under conditions foreffective production and recovery of the protein. The nucleic acid canbe operably linked to a heterologous promoter, e.g., an induciblepromoter or a constitutive promoter. Effective growth conditions aretypically, but not necessarily, in liquid media comprising salts, water,carbon, nitrogen, phosphate sources, minerals, and other nutrients, but.may be any solution in which PANZP proteins may be produced.

In one embodiment, recovery of the protein may refer to collecting thegrowth solution and need not involve additional steps of purification.Proteins of the present invention, however, can be purified usingstandard purification techniques, such as, but not limited to, affinitychromatography, thermaprecipitation, immunoaffinity chromatography,ammonium sulfate precipitation, ion exchange chromatography, filtration,electrophoresis, hydrophobic interaction chromatography, and others.

The PAN and ZP domain containing receptor-like polypeptide can be fusedto an affinity tag, e.g., a purification handle (e.g.,glutathione-S-reductase, hexa-histidine, maltose binding protein,dihydrofolate reductases, or chitin binding protein) or an epitope tag(e.g., c-myc epitope tag, FLAG™ tag, or influenza HA tag). Affinitytagged and epitope tagged proteins can be purified using routineart-known methods.

Antibodies Against PAN and ZP Domain Containing Receptor-LikePolypeptides

Recombinant PAN and ZP domain containing receptor-like gene products orderivatives thereof can be used to produce immunologically interactivemolecules, such as antibodies, or functional derivatives thereof. Usefulantibodies include those that bind to a polypeptide that hassubstantially the same sequence as the amino acid sequences recited inSEQ ID NO: 3, 4, 10, 11 and/or 12, or that has at least 80% similarityover 50 or more amino acids to these sequences. In a preferredembodiment, the antibody specifically binds to a polypeptide having theamino acid sequence recited in SEQ ID NO: 3, 4, 10, 11 and/or 12. Theantibodies can be antibody fragments and genetically engineeredantibodies, including single chain antibodies or chimeric antibodiesthat can bind to more than one epitope. Such antibodies may bepolyclonal or monoclonal and may be selected from naturally occurringantibodies or may be specifically raised to a recombinant PANZP protein.

Antibodies can be derived by immunization with a recombinant or purifiedPANZP gene or gene product. As used herein, the term “antibody” refersto an immunoglobulin, or fragment thereof. Examples of antibodyfragments include F(ab) and F(ab′)₂ fragments, particularly functionalones able to bind epitopes. Such fragments can be generated byproteolytic cleavage, e.g., with pepsin, or by genetic engineering.Antibodies can be polyclonal, monoclonal, or recombinant. In addition,antibodies can be modified to be chimeric, or humanized. Further, anantibody can be coupled to a label or a toxin.

Antibodies can be generated against a full-length PANZP protein, or afragment thereof, e.g., an antigenic peptide. Such polypeptides can becoupled to an adjuvant to improve immunogenicity. Polyclonal serum isproduced by injection of the antigen into a laboratory animal such as arabbit and subsequent collection of sera. Alternatively, the antigen isused to immunize mice. Lymphocytic cells are obtained from the mice andfused with myelomas to form hybridomas producing antibodies.

Peptides for generating PAN and ZP domain containing receptor-likeantibodies can be about 8, 10, 15, 20, 30 or more amino acid residues inlength, e.g., a peptide of such length obtained from SEQ ID NO: 3, 4,10, 11 and/or 12. Useful peptides include those containing a PAN or ZPdomain, e.g., a PAN or ZP domain listed in Table 3. Peptides or epitopescan also be selected from regions exposed on the surface of the protein,e.g., hydrophilic or amphipathic regions. An epitope in the vicinity ofan active or binding site can be selected such that an antibody bindingsuch an epitope would block access to the active site or preventbinding. Antibodies reactive with, or specific for, any of theseregions, or other regions or domains described herein are provided. Anantibody to a PANZP protein can modulate a PANZP binding activity.

Monoclonal antibodies, which can be produced by routine methods, areobtained in abundance and in homogenous form from hybridomas formed fromthe fusion of immortal cell lines (e.g., myelomas) with lymphocytesimmunized with PANZP polypeptides such as those set forth in SEQ ID NO:3, 4, 10, 11 and/or 12.

In addition, antibodies can be engineered, e.g., to produce a singlechain antibody (see, for example, Colcher et al. (1999) Ann N YAcad Sci880: 263-280; and Reiter (1996) Clin Cancer Res 2: 245-252). In stillanother implementation, antibodies are selected or modified based onscreening procedures, e.g., by screening antibodies or fragments thereoffrom a phage display library.

Antibodies of the present invention have a variety of important useswithin the scope of this invention. For example, such antibodies can beused: (i) as therapeutic compounds to passively immunize an animal inorder to protect the animal from nematodes susceptible to antibodytreatment; (ii) as reagents in experimental assays to detect presence ofnematodes; (iii) as tools to screen for expression of the gene productin nematodes, animals, fungi, bacteria, and plants; and/or (iv) as apurification tool of PANZP protein; (v) as PANZP inhibitors/activatorsthat can be expressed or introduced into plants or animals fortherapeutic purposes.

An antibody against a PAN and ZP domain containing receptor-like proteincan be produced in a plant cell, e.g., in a transgenic plant or inculture (see, e.g., U.S. Pat. No. 6,080,560).

Antibodies that specifically recognize a S. stercoralis, M. javanica, H.glycines, and/or B. malayi PANZP proteins can be used to identify S.stercoralis, M. javanica, H. glycines, and/or B. malayi nematodes, and,thus, can be used to diagnose and/or monitor a disease caused by S.stercoralis, M javanica, H. glycines, and/or B. malayi.

Immunization

The PANZP proteins of the invention and fragments thereof (e.g., afragment that includes one or more PAN or ZP domains) can be used toimmunize a mammal, e.g., a human, primate, or dog. The protein orpeptide fragment can be introduced into a mammal as a unit dose inoculumin combination with any physiologically suitable diluent. One or moreinoculums can be administered. Each inoculum can contain an amount ofpolypeptide effective to elicit an immune response, preferably aprotective immune response that reduces the occurrence of subsequentinfection by a nematode, e.g., S. steroralis or B. malayi. A unit dosecan contain, e.g., at least 0.1, preferably at least 0.5 milligrams/kgof body weight of host.

The PANZP peptide immunogen can contain 10, 20, 30, 50, 100 or moreamino acids and can include all or part of a PAN or ZP domain, e.g., aPAN or ZP domain listed in Table 3. The PANZP peptide immunogen caninclude 2, 3, 4, or more PANZP peptides that are the same or different.Moreover, the PANZP peptides can be flanked by other amino acidsequences. Thus, the immunogen can contain, e.g., two copies of a givenPAN domain separated by a linker. The immunogen can include one or moreportions of one, two or more PANZP proteins. Thus, the immunogen caninclude a portion of S. steroralis or B. malayi PANZP1 and a portion ofS. steroralis PANZP2. The inoculum can include two or morenon-contiguous portions of a PANZP protein, e.g., two or more portionsincluding PAN domains.

The inoculum can include an adjuvant, e.g., complete .or incompleteFreund's adjuvant. The PANZP peptide can be linked to a carrier such astetanus toxoid, human BSA, or KLH. The inoculum can include stabilizers(e.g., sugars, preservatives, wetting agents, emulsifying agents,buffering agents, dyes, and additives) that improve viscosity ofsyringability. The inoculum can be administered once or multiple times(e.g., a prime and a boost).

A mammal can be inoculated by intravenous, intraperitoneal, intradermal,subcutaneous, or intramuscular method. Inoculation can be via a needleor needleless means.

Nucleic Acids Agents

Also featured are isolated nucleic acids that are antisense to nucleicacids encoding nematode PAN and ZP domain containing receptor-likeproteins. An “antisense” nucleic acid includes a sequence that iscomplementary to the coding strand of a nucleic acid encoding a PANZPprotein. The complementarity can be in a coding region of the codingstrand or in a noncoding region, e.g., a 5′- or 3′-untranslated region,e.g., the translation start site. The antisense nucleic acid can beproduced from a cellular promoter (e.g., a RNA polymerase II or IIIpromoter), or can be introduced into a cell, e.g., using a liposome. Forexample, the antisense nucleic acid can be a synthetic oligonucleotidehaving a length of about 10, 15, 20, 30, 40, 50, 75, 90, 120 or morenucleotides in length.

An antisense nucleic acid can be synthesized chemically or producedusing enzymatic reagents, e.g., a ligase. An antisense nucleic acid canalso incorporate modified nucleotides, and artificial backbonestructures, e.g., phosphorothioate derivative, and acridine substitutednucleotides.

Ribozmes

The antisense nucleic acid can be a ribozyme. The ribozyme can bedesigned to specifically cleave RNA, e.g., a PANZP mRNA. Methods fordesigning such ribozymes are described in U.S. Pat. No. 5,093,246 orHaselhoff and Gerlach (1988) Nature 334:585-591. For example, theribozyme can be a derivative of Tetrahymena L-19 IVS RNA in which thenucleotide sequence of the active site is modified to be complementaryto a PANZP nucleic acid (see, e.g., Cech et al. U.S. Pat. No. 4,987,071;and Cech et al. U.S. Pat. No. 5,116,742).

Peptide Nucleic acid (PNA)

An antisense agent directed against an PAN and ZP domain containingreceptor-like nucleic acid can be a peptide nucleic acid (PNA). SeeHyrup et al. (1996) Bioorganic & Medicinal Chemistry 4: 5-23) formethods and a description of the replacement of the deoxyribosephosphate backbone for a pseudopeptide backbone. A PNA can specificallyhybridize to DNA and RNA under conditions of low ionic strength as aresult of its electrostatic properties. The synthesis of PNA oligomerscan be performed using standard solid phase peptide synthesis protocolsas described in Hyrup et al. (1996) supra and Perry-O'Keefe et al. Proc.Natl. Acad. Sci. 93: 14670-14675.

RNA Mediated Interference (RNAI)

A double stranded RNA (dsRNA) molecule can be used to inactivate a PANand ZP domain containing receptor-like gene in a cell by a process knownas RNA mediated-interference (RNAi; e.g., Fire et al. (1998) Nature391:806-811, and Gönczy et al. (2000) Nature 408:331-336). The dsRNAmolecule can have the nucleotide sequence of a PANZP nucleic aciddescribed herein or a fragment thereof. The molecule can be injectedinto a cell, or a syncytium, e.g., a nematode gonad as described in Fireet al., supra. Alternatively, the molecule can be used to eradicate anematode infection in vertebrates or other animals by delivery to anematode-infected animal by injection or oral dosing.

Transgenic RNAi

A double stranded RNA (dsRNA) molecule can be used to inactivate a PANand ZP domain containing receptor-like gene in a cell by a process knownas RNA mediated-interference (RNAi; e.g., Fire et al. (1998) Nature391:806-811, and Gönczy et al. (2000) Nature 408:331-336). The dsRNAmolecule can have the nucleotide sequence of all or a portion of a PANZPnucleic acid described herein or a fragment thereof. The RNAi triggeringmolecule can be produced by a transgenic plant engineered to producedsRNA homologous to a PAN ZP domain-containing receptor-like gene anddelivered to a plant parasitic nematode when it attacks and/or feeds onthe transgenic plant. Various techniques are known in the art forexpressing in plants nucleic acid molecule that inactivate a selectedgene, including a nematode gene via RNAi or a related mechanism (see,e.g., Boutla et al. (2002) Nucl. Acids Res. 30:1688; and Wesley et al.(2001) Plant J. 27:581).

Screening Assays

Another embodiment of the present invention is a method of identifying acompound capable of altering (e.g., inhibiting or enhancing) theactivity of PANZP molecules. This method, also referred to as a“screening assay,” herein, includes, but is not limited to, thefollowing procedure: (i) contacting an isolated PANZP protein (or aportion thereof, e.g., a PAN or ZP domain) with a test inhibitorycompound under conditions in which, in the absence of the test compound,the protein has PANZP activity; and (ii) determining if the testcompound alters the PANZP activity (i.e., binding of PANZP to itssubstrates). Suitable inhibitors or activators that alter a nematodePANZP activity include compounds that interface directly with a nematodePANZP protein substrate binding interaction. Compounds can also interactwith other regions of the nematode PANZP protein outside the bindinginterface and enhance or interfere with PANZP-substrate interactions(e.g., allosteric effects).

Compounds

A test compound can be a large or small molecule, for example, anorganic compound with a molecular weight of about 100 to 10,000; 200 to5,000; 200 to 2000; or 200 to 1,000 daltons. A test compound can be anychemical compound, for example, a small organic molecule, acarbohydrate, a lipid, an amino acid, a polypeptide, a nucleoside, anucleic acid, or a peptide nucleic acid. Small molecules include, butare not limited to, metabolites, metabolic analogues, peptides,peptidomimetics (e.g., peptoids), amino acids, amino acid analogs,polynucleotides, polynucleotide analogs, nucleotides, nucleotideanalogs, organic or inorganic compounds (i.e., including heteroorganicand organometallic compounds). Compounds and components for synthesis ofcompounds can be obtained from a commercial chemical supplier, e.g.,Sigma-Aldrich Corp. (St. Louis, Mo.). The test compound or compounds canbe naturally occurring, synthetic, or both. A test compound can be theonly substance assayed by the method described herein. Alternatively, acollection of test compounds can be assayed either consecutively orconcurrently by the methods described herein.

Compounds can act by allosteric inhibition or by directly by preventingthe substrate PANZP interaction.

A high-throughput method can be used to screen large libraries ofchemicals. Such libraries of candidate compounds can be generated orpurchased, e.g., from Chembridge Corp. (San Diego, Calif.). Librariescan be designed to cover a diverse range of compounds. For example, alibrary can include 10,000, 50,000, or 100,000 or more unique compounds.Merely by way of illustration, a library can be constructed fromheterocycles including pyridines, indoles, quinolines, furans,pyrimidines, triazines, pyrroles, imidazoles, naphthalenes,benzimidazoles, piperidines, pyrazoles, benzoxazoles, pyrrolidines,thiphenes, thiazoles, benzothiazoles, and morpholines. A library can bedesigned and synthesized to cover such classes of chemicals, e.g., asdescribed in DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A.90:6909-6913; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422-11426; Zuckermann et al. (1994) J. Med. Chem. 37:2678-2685; Choet al. (1993) Science 261:1303-1305; Carrell et al. (1994) Angew. Chem.Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl.33:2061; and Gallop et al. (1994) J. Med. Chem. 37:1233-1251.

Organism-based Assays

Organisms can be grown in microtiter plates, e.g., 6-well, 32-well,64-well, 96-well, 384-well plates.

In one embodiment, the organism is a nematode. The nematodes can begenetically modified. Non-limiting examples of such modified nematodesinclude: 1) nematodes or nematode cells (S. stercoralis. M. javanica, H.glycines, B. malayi and/or C. elegans) having one or more PANZP genesinactivated (e.g., using RNA mediated interference); 2) nematodes ornematode cells expressing a heterologous PANZP gene, e.g., an PANZP genefrom another species; and 3) nematodes or nematode cells having one ormore endogenous PANZP genes inactivated and expressing a heterologousPANZP gene, e.g., a S. stercoralis. M. javanica, H. glycines, B. malayiand/or C. elegans PANZP gene as described herein.

A plurality of candidate compounds, e.g., a combinatorial library, canbe screened. The library can be provided in a format that is amenablefor robotic manipulation, e.g., in microtitre plates. Compounds can beadded to the wells of the microtiter plates. Following compound additionand incubation, viability and/or reproductive properties of thenematodes or nematode cells are monitored.

The compounds can also be pooled, and the pools tested. Positive poolsare split for subsequent analysis. Regardless of the method, compoundsthat decrease the viability or reproductive ability of nematodes,nematode cells, or progeny of the nematodes are considered leadcompounds.

In another embodiment, the compounds can be tested on a microorganism ora eukaryotic or mammalian cell line, e.g., rabbit skin cells, Chinesehamster ovary cells (CHO), and/or Hela cells. For example, CHO cellsabsent for PANZP genes, but expressing a nematode PANZP gene can beused. The generation of such strains is routine in the art. As describedabove for nematodes and nematode cells, the cell lines can be grown inmicrotitre plates, each well having a different candidate compound orpool of candidate compounds. Growth is monitored during or after theassay to determine if the compound or pool of compounds is a modulatorof a nematode PANZP polypeptide.

In Vitro Binding Assays

The screening assay can also be a cell-free binding assay, e.g., anassay to identify compounds that bind a nematode PANZP polypeptide. Forexample, a nematode PANZP polypeptide can be purified and labeled. Thelabeled polypeptide is contacted to beads; each bead has a tagdetectable by mass spectroscopy, and test compound, e.g., a compoundsynthesized by combinatorial chemical methods. Beads to which thelabeled polypeptide is bound are identified and analyzed by massspectroscopy. The beads can be generated using “split-and-pool”synthesis. The method can further include a second assay to determine ifthe compound alters the activity of the PANZP polypeptide.

Optimization of a Compound

Once a lead compound has been identified, standard principles ofmedicinal chemistry can be used to produce derivatives of the compound.Derivatives can be screened for improved pharmacological properties, forexample, efficacy, pharmacokinetics, stability, solubility, andclearance. The moieties responsible for a compound's activity in theabove-described assays can be delineated by examination ofstructure-activity relationships (SAR) as is commonly practiced in theart. One can modify moieties on a lead compound and measure the effectsof the modification on the efficacy of the compound to thereby producederivatives with increased potency. For an example, see Nagarajan et al.(1988) J. Antibiot. 41:1430-1438. A modification can includeN-acylation, amination, amidation, oxidation, reduction, alkylation,esterification, and hydroxylation. Furthermore, if the biochemicaltarget of the lead compound is known or determined, the structure of thetarget and the lead compound can inform the design and optimization ofderivatives. Molecular modeling software to do this is commerciallyavailable (e.g., Molecular Simulations, Inc.). “SAR by NMR,” asdescribed in Shuker et al. (1996) Science 274:1531-1534, can be used todesign ligands with increased affinity, by joining lower-affinityligands.

A preferred compound is one that interferes with the function of anematode PAN and ZP domain containing receptor-like polypeptide and thatis not substantially toxic to plants, animals, or humans. By “notsubstantially toxic” it is meant that the compound does notsubstantially affect the activity of animal, or human PAN or ZPcontaining proteins. Thus, particularly desirable inhibitors of S.stercoralis. M. javanica, H. glycines, B. malayi and/or C. elegans PANZPdo not substantially inhibit human plasminogen, hepatycyte growthfactor, Factor XI, or uromodulin activity of vertebrates, e.g., humansfor example.

Standard pharmaceutical procedures can be used to assess the toxicityand therapeutic efficacy of a modulator of a PANZP activity. The LD50(the dose lethal to 50% of the population) and the ED50 (the dosetherapeutically effective in 50% of the population can be measured incell cultures, experimental plants (e.g., in laboratory or fieldstudies), or experimental animals. Optionally, a therapeutic index canbe determined which is expressed as the ratio: LD50/ED50. Hightherapeutic indices are indicative of a compound being an effectivePANZP inhibitor, while not causing undue toxicity or side effects to asubject (e.g., a host plant or host animal).

Alternatively, the ability of a candidate compound to modulate anon-nematode PAN or ZP containing polypeptide is assayed, e.g., by amethod described herein. For example, the binding affinity of acandidate compound for a mammalian PAN containing polypeptide can bemeasured and compared to the binding affinity for a nematode PANZPpolypeptide.

The aforementioned analyses can be used to identify and/or design amodulator with specificity for nematode PAN and ZP domain containingreceptor-like polypeptide over vertebrate or other animal (e.g.,mammalian) PAN or ZP containing polypeptides. Suitable nematodes totarget are any nematodes with the PANZP proteins or proteins that can betargeted by compounds that otherwise inhibit, reduce, activate, orgenerally effect the activity of nematode PANZP proteins.

Inhibitors of nematode PAN and ZP domain containing receptor-likeproteins can also be used to identify PAN and ZP domain containingreceptor-like proteins in the nematode or other organisms usingprocedures known in the art, such as affinity chromatography. Forexample, a specific antibody may be linked to a resin and a nematodeextract passed over the resin, allowing any PANZP proteins that bind theantibody to bind the resin. Subsequent biochemical techniques familiarto those skilled in the art can be performed to purify and identifybound PANZP proteins.

Agricultural Compositions

A compound that is identified as a PAN and ZP domain containingreceptor-like polypeptide inhibitor can be formulated as a compositionthat is applied to plants, soil, or seeds in order to confer nematoderesistance. The composition can be prepared in a solution, e.g., anaqueous solution, at a concentration from about 0.005% to 10%, or about0.01% to 1%, or about 0.1% to 0.5% by weight. The solution can includean organic solvent, e.g., glycerol or ethanol. The composition can beformulated with one or more agriculturally acceptable carriers.Agricultural carriers can include: clay, talc, bentonite, diatomaceousearth, kaolin, silica, benzene, xylene, toluene, kerosene,N-methylpyrrolidone, alcohols (methanol, ethanol, isopropanol,n-butanol, ethylene glycol, propylene glycol, and the like), and ketones(acetone, methylethyl ketone, cyclohexanone, and the like). Theformulation can optionally further include stabilizers, spreadingagents, wetting extenders, dispersing agents, sticking agents,disintegrators, and other additives, and can be prepared as a liquid, awater-soluable solid (e.g., tablet, powder or granule), or a paste.

Prior to application, the solution can be combined with another desiredcomposition such as another anthelmintic agent, germicide, fertilizer,plant growth regulator and the like. The solution may be applied to theplant tissue, for example, by spraying, e.g., with an atomizer, bydrenching, by pasting, or by manual application, e.g., with a sponge.The solution can also be distributed from an airborne source, e.g., anaircraft or other aerial object, e.g., a fixture mounted with anapparatus for spraying the solution, the fixture being of sufficientheight to distribute the solution to the desired plant tissues.Alternatively, the composition can be applied to plant tissue from avolatile or airborne source. The source is placed in the vicinity of theplant tissue and the composition is dispersed by diffusion through theatmosphere. The source and the plant tissue to be contacted can beenclosed in an incubator, growth chamber, or greenhouse, or can be insufficient proximity that they can be outdoors.

If the composition is distributed systemically thorough the plant, thecomposition can be applied to tissues other than the leaves, e.g., tothe stems or roots. Thus, the composition can be distributed byirrigation. The composition can also be injected directly into roots orstems.

A skilled artisan would be able to determine an appropriate dosage forformulation of the active ingredient of the composition. For example,the ED50 can be determined as described above from experimental data.The data can be obtained by experimentally varying the dose of theactive ingredient to identify a dosage effective for killing a nematode,while not causing toxicity in the host plant or host animal (i.e.non-nematode animal).

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. An isolated nucleic acid molecule comprising a nucleotide sequenceencoding the amino acid sequence of SEQ ID NO:10.
 2. An isolated nucleicacid molecule comprising a nucleotide sequence encoding a polypeptidecomprising an amino acid sequence that is at least 95% identical to theamino acid sequence of SEQ ID NO:10, wherein the ploypeptide comprisessix PAN domains ans one ZP domain and is a secreted membrane-boundprotein.
 3. An isolated nucleic acid molecule comprising the nucleotidesequence of SEQ ID NO:13.
 4. An isolated nucleic acid moleculeconsisting of the nucleotide sequence of SEQ ID NO:13.
 5. A vectorcomprising the isolated nucleic acid molecule of any of claims 1, 2, 3and 4.